Alzheimer&#39;s disease secretase, APP substrates therefor, and uses therefor

ABSTRACT

The present invention provides the enzyme and enzymatic procedures for cleaving the β secretase cleavage site of the APP protein and associated nucleic acids, peptides, vectors, cells and cell isolates and assays. The invention further provides a modified APP protein and associated nucleic acids, peptides, vectors, cells, and cell isolates, and assays that are particularly useful for identifying candidate therapeutics for treatment or prevention of Alzheimer&#39;s disease.

This is a divisional application of U.S. application Ser. No.09/416,901, filed on Oct. 13, 1999, which claims priority benefit ofU.S. Provisional Patent Application No. 60/155,493, filed Sep. 23, 1999.The present application also claims priority benefit as acontinuation-in-part of U.S. patent application Ser. No. 09/404,133 andPCT/US99/20881, both filed Sep. 23, 1999, both of which in turn claimpriority benefit of U.S. Provisional Patent Application No. 60/101,594,filed Sep. 24, 1998. All of these priority applications are herebyincorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to Alzheimer's Disease, amyloid proteinprecursor, amyloid beta peptide, and human aspartyl proteases, as wellas a method for the identification of agents that modulate the activityof these polypeptides and thereby are candidates to modulate theprogression of Alzheimer's disease.

BACKGROUND OF THE INVENTION

Alzheimer's disease (AD) causes progressive dementia with consequentformation of amyloid plaques, neurofibrillary tangles, gliosis andneuronal loss. The disease occurs in both genetic and sporadic formswhose clinical course and pathological features are quite similar. Threegenes have been discovered to date which, when mutated, cause anautosomal dominant form of Alzheimer's disease. These encode the amyloidprotein precursor (APP) and two related proteins, presenilin-1 (PS1) andpresenilin-2 (PS2), which, as their names suggest, are structurally andfunctionally related. Mutations in any of the three proteins have beenobserved to enhance proteolytic processing of APP via an intracellularpathway that produces amyloid beta peptide (Aβ peptide ,or sometimeshere as Abeta), a 40-42 amino acid long peptide that is the primarycomponent of amyloid plaque in AD.

Dysregulation of intracellular pathways for proteolytic processing maybe central to the pathophysiology of AD. In the case of plaqueformation, mutations in APP, PS1 or PS2 consistently alter theproteolytic processing of APP so as to enhance formation of Aβ 1-42, aform of the Aβ peptide which seems to be particularly amyloidogenic, andthus very important in AD. Different forms of APP range in size from695-770 amino acids, localize to the cell surface, and have a singleC-terminal transmembrane domain. Examples of specific isotypes of APPwhich are currently known to exist in humans are the 695-amino acidpolypeptide described by Kang et. al. (1987), Nature 325: 733-736 whichis designated as the “normal” APP; the 751 amino acid polypeptidedescribed by Ponte et al. (1988), Nature 331: 525-527 (1988) and Tanziet al. (1988), Nature 331: 528-530; and the 770 amino acid polypeptidedescribed by Kitaguchi et. al. (1988), Nature 331: 530-532. The Abetapeptide is derived from a region of APP adjacent to and containing aportion of the transmembrane domain. Normally, processing of APP at theα-secretase site cleaves the midregion of the Aβ sequence adjacent tothe membrane and releases the soluble, extracellular domain of APP fromthe cell surface. This α-secretase APP processing creates soluble APP-α,which is normal and not thought to contribute to AD. Pathologicalprocessing of APP at the β- and γ-secretase sites, which are locatedN-terminal and C-terminal to the α-secretase site, respectively,produces a very different result than processing at the α site.Sequential processing at the β- and γ-secretase sites releases the Aβpeptide, a peptide possibly very important in AD pathogenesis.Processing at the β- and γ-secretase sites can occur in both theendoplasmic reticulum (in neurons) and in the endosomal/lysosomalpathway after reinternalization of cell surface APP (in all cells).Despite intense efforts, for 10 years or more, to identify the enzymesresponsible for processing APP at the β and γ sites, to produce the Aβpeptide, those proteases remained unknown until this disclosure.

SUMMARY OF THE INVENTION

Here, for the first time, we report the identification andcharacterization of the β secretase enzyme, termed Aspartyl Protease 2(Asp2). We disclose some known and some novel human aspartic proteasesthat can act as β-secretase proteases and, for the first time, weexplain the role these proteases have in AD. We describe regions in theproteases critical for their unique function and for the first timecharacterize their substrate. This is the first description of expressedisolated purified active protein of this type, assays that use theprotein, in addition to the identification and creation of useful celllines and inhibitors.

Here we disclose a number of variants of the Asp2 gene and peptide.

In one aspect, the invention provides any isolated or purified nucleicacid polynucleotide that codes for a protease capable of cleaving thebeta (β) secretase cleavage site of APP that contains two or more setsof special nucleic acids, where the special nucleic acids are separatedby nucleic acids that code for about 100 to 300 amino acid positions,where the amino acids in those positions may be any amino acids, wherethe first set of special nucleic acids consists of the nucleic acidsthat code for the peptide DTG, where the first nucleic acid of the firstspecial set of nucleic acids is the first special nucleic acid, andwhere the second set of nucleic acids code for either the peptide DSG orDTG, where the last nucleic acid of the second set of nucleic acids isthe last special nucleic acid, with the proviso that the nucleic acidsdisclosed in SEQ ID NO. 1 and SEQ ID NO. 3 are not included. In apreferred embodiment, the two sets of special nucleic acids areseparated by nucleic acids that code for about 125 to 222 amino acidpositions, which may be any amino acids. In a highly preferredembodiment, the two sets of special nucleic acids are separated bynucleic acids that code for about 150 to 196, or 150-190, or 150 to 172amino acid positions, which may be any amino acids. In a particularpreferred embodiment, the two sets are separated by nucleic acids thatcode for about 172 amino acid positions, which may be any amino acids.An exemplary nucleic acid polynucleotide comprises the acid nucleotidesequence in SEQ ID NO. 5. In another particular preferred embodiment,the two sets are separated by nucleic acids that code for about 196amino acids. An exemplary polynucleotide comprises the nucleotidesequence in SEQ ID NO. 5. In another particular embodiment, the two setsof nucleotides are separated by nucleic acids that code for about 190amino acids. An exemplary polynucleotide comprises the nucleotidesequence in SEQ ID NO. 1. Preferably, the first nucleic acid of thefirst special set of amino acids, that is, the first special nucleicacid, is operably linked to any codon where the nucleic acids of thatcodon codes for any peptide comprising from 1 to 10,000 amino acid(positions). In one variation, the first special nucleic acid isoperably linked to nucleic acid polymers that code for any peptideselected from the group consisting of: any reporter proteins or proteinswhich facilitate purification. For example, the first special nucleicacid is operably linked to nucleic acid polymers that code for anypeptide selected from the group consisting of: immunoglobin-heavy chain,maltose binding protein, glutathione S transferase, Green Fluorescentprotein, and ubiquitin. In another variation, the last nucleic acid ofthe second set of special amino acids, that is, the last special nucleicacid, is operably linked to nucleic acid polymers that code for anypeptide comprising any amino acids from 1 to 10,000 amino acids. Instill another variation, the last special nucleic acid is operablylinked to nucleic acid polymers that code for any peptide selected fromthe group consisting of: any reporter proteins or proteins whichfacilitate purification. For example, the last special nucleic acid isoperably linked to nucleic acid polymers that code for any peptideselected from the group consisting of: immunoglobin-heavy chain, maltosebinding protein, glutathione S transferase, Green Fluorescent protein,and ubiquitin.

In a related aspect, the invention provides any isolated or purifiednucleic acid polynucleotide that codes for a protease capable ofcleaving the beta secretase cleavage site of APP that contains two ormore sets of special nucleic acids, where the special nucleic acids areseparated by nucleic acids that code for about 100 to 300 amino acidpositions, where the amino acids in those positions may be any aminoacids, where the first set of special nucleic acids consists of thenucleic acids that code for DTG, where the first nucleic acid of thefirst special set of nucleic acids is the first special nucleic acid,and where the second set of nucleic acids code for either DSG or DTG,where the last nucleic acid of the second set of special nucleic acidsis the last special nucleic acid, where the first special nucleic acidis operably linked to nucleic acids that code for any number of aminoacids from zero to 81 amino acids and where each of those codons maycode for any amino acid. In a preferred embodiment, the first specialnucleic acid is operably linked to nucleic acids that code for anynumber of from 64 to 77 amino acids where each codon may code for anyamino acid. In a particular embodiment, the first special nucleic acidis operably linked to nucleic acids that code for 71 amino acids. Forexample, the first special nucleic acid is operably linked to 71 aminoacids and where the first of those 71 amino acids is the amino acid T.In a preferred embodiment, the polynucleotide comprises a sequence thatis at least 95% identical to a human Asp1 or Asp2 sequence as taughtherein. In another preferred embodiment, the first special nucleic acidis operably linked to nucleic acids that code for any number of from 30to 54 amino acids, or 35 to 47 amino acids, or 40 to 54 amino acidswhere each codon may code for any amino acid. In a particularembodiment, the first special nucleic acid is operably linked to nucleicacids that code for 47 amino acids. For example, the first specialnucleic acid is operably linked to 47 codons where the first those 47amino acids is the amino acid E.

In another related aspect, the invention provides for any isolated orpurified nucleic acid polynucleotide that codes for a protease capableof cleaving the beta (β) secretase cleavage site of APP and thatcontains two or more sets of special nucleic acids, where the specialnucleic acids are separated by nucleic acids that code for about 100 to300 amino acid positions, where the amino acids in those positions maybe any amino acids, where the first set of special nucleic acidsconsists of the nucleic acids that code for the peptide DTG, where thefirst nucleic acid of the first special set of amino acids is, the firstspecial nucleic acid, and where the second set of special nucleic acidscode for either the peptide DSG or DTG, where the last nucleic acid ofthe second set of special nucleic acids, the last special nucleic acid,is operably linked to nucleic acids that code for any number of codonsfrom 50 to 170 codons. In a preferred embodiment, the last specialnucleic acid is operably linked to nucleic acids comprising from 100 to170 codons. In a highly preferred embodiment, the last special nucleicacid is operably linked to nucleic acids comprising from 142 to 163codons. In a particular embodiment, the last special nucleic acid isoperably linked to nucleic acids comprising about 142 codons, or about163 codons, or about 170 codons. In a highly preferred embodiment, thepolynucleotide comprises a sequence that is at least 95% identical toaspartyl-protease encoding sequences taught herein. In one variation,the second set of special nucleic acids code for the peptide DSG. Inanother variation, the first set of nucleic acid polynucleotide isoperably linked to a peptide purification tag. For example, the nucleicacid polynucleotide is operably linked to a peptide purification tagwhich is six histidine. In still another variation, the first set ofspecial nucleic acids are on one polynucleotide and the second set ofspecial nucleic acids are on a second polynucleotide, where both firstand second polynucleotides have at lease 50 codons. In one embodiment ofthis type, both of the polynucleotides are in the same solution. In arelated aspect, the invention provides a vector which contains apolynucleotide as described above, or a cell or cell line which istransformed or transfected with a polynucleotide as described above orwith a vector containing such a polynucleotide.

In still another aspect, the invention provides an isolated or purifiedpeptide or protein comprising an amino acid polymer that is a proteasecapable of cleaving the beta (β) secretase cleavage site of APP thatcontains two or more sets of special amino acids, where the specialamino acids are separated by about 100 to 300 amino acid positions,where each amino acid position can be any amino acid, where the firstset of special amino acids consists of the peptide DTG, where the firstamino acid of the first special set of amino acids is, the first specialamino acid, where the second set of amino acids is selected from thepeptide comprising either DSG or DTG, where the last amino acid of thesecond set of special amino acids is the last special amino acid, withthe proviso that the proteases disclosed in SEQ ID NO. 2 and SEQ ID NO.4 are not included. In preferred embodiments, the two sets of aminoacids are separated by about 125 to 222 amino acid positions or about150 to 196 amino acids, or about 150-190 amino acids, or about 150 to172 amino acids, where in each position it may be any amino acid. In aparticular embodiment, the two sets of amino acids are separated byabout 172 amino acids. For example, the protease has the amino acidsequence described in SEQ ID NO 6. In another particular embodiment, thetwo sets of amino acids are separated by about 196 amino acids. Forexample, the two sets of amino acids are separated by the same aminoacid sequences that separate the same set of special amino acids in SEQID NO 4. In another particular embodiment, the two sets of nucleotidesare separated by about 190 amino acids. For example, the two sets ofnucleotides are separated by the same amino acid sequences that separatethe same set of special amino acids in SEQ ID NO 2. In one embodiment,the first amino acid of the first special set of amino acids, that is,the first special amino acid, is operably linked to any peptidecomprising from 1 to 10,000 amino acids. In another embodiment, thefirst special amino acid is operably linked to any peptide selected fromthe group consisting of: any reporter proteins or proteins whichfacilitate purification. In particular embodiments, the first specialamino acid is operably linked to any peptide selected from the groupconsisting of: immunoglobin-heavy chain, maltose binding protein,glutathione S transferase, Green Fluorescent protein, and ubiquitin. Instill another variation, the last amino acid of the second set ofspecial amino acids, that is, the last special amino acid, is operablylinked to any peptide comprising any amino acids from 1 to 10,000 aminoacids. By way of nonlimiting example, the last special amino acid isoperably linked any peptide selected from the group consisting of anyreporter proteins or proteins which facilitate purification. Inparticular embodiments, the last special amino acid is operably linkedto any peptide selected from the group consisting of: immunoglobin-heavychain, maltose binding protein, glutathione S transferase, GreenFluorescent protein, and ubiquitin.

In a related aspect, the invention provides any isolated or purifiedpeptide or protein comprising an amino acid polypeptide that codes for aprotease capable of cleaving the beta secretase cleavage site of APPthat contains two or more sets of special amino acids, where the specialamino acids are separated by about 100 to 300 amino acid positions,where each amino acid in each position can be any amino acid, where thefirst set of special amino acids consists of the amino acids DTG, wherethe first amino acid of the first special set of amino acids is, thefirst special amino acid, D, and where the second set of amino acids iseither DSG or DTG, where the last amino acid of the second set ofspecial amino acids is the last special amino acid, G, where the firstspecial amino acid is operably linked to amino acids that code for anynumber of amino acids from zero to 81 amino acid positions where in eachposition it may be any amino acid. In a preferred embodiment, the firstspecial amino acid is operably linked to a peptide from about 30-77 orabout 64 to 77 amino acids positions where each amino acid position maybe any amino acid. In a particular embodiment, the first special aminoacid is operably linked to a peptide 35, 47, 71, or 77 amino acids. In avery particular embodiment, the first special amino acid is operablylinked to 71 amino acids and the first of those 71 amino acids is theamino acid T. For example, the polypeptide comprises a sequence that isat least 95% identical to an aspartyl protease sequence as describedherein. In another embodiment, the first special amino acid is operablylinked to any number of from 40 to 54 amino acids (positions) where eachamino acid position may be any amino acid. In a particular embodiment,the first special amino acid is operably linked to amino acids that codefor a peptide of 47 amino acids. In a very particular embodiment, thefirst special amino acid is operably linked to a 47 amino acid peptidewhere the first those 47 amino acids is the amino acid E. In anotherparticular embodiment, the first special amino acid is operably linkedto the same corresponding peptides from SEQ ID NO. 3 that are 35, 47,71, or 77 peptides in length, beginning counting with the amino acids onthe first special sequence, DTG, towards the N-terminal of SEQ ID NO. 3.In another particular embodiment, the polypeptide comprises a sequencethat is at least 95% identical to the same corresponding amino acids inSEQ ID NO. 4, that is, identical to that portion of the sequences in SEQID NO. 4, including all the sequences from both the first and or thesecond special nucleic acids, toward the—terminal, through and including71, 47, 35 amino acids before the first special amino acids. Forexample, the complete polypeptide comprises the peptide of 71 aminoacids, where the first of the amino acid is T and the second is Q.

In still another related aspect, the invention provides any isolated orpurified amino acid polypeptide that is a protease capable of cleavingthe beta (β) secretase cleavage site of APP that contains two or moresets of special amino acids, where the special amino acids are separatedby about 100 to 300 amino acid positions, where each amino acid in eachposition can be any amino acid, where the first set of special aminoacids consists of the amino acids that code for DTG, where the firstamino acid of the first special set of amino acids is, the first specialamino acid, D, and where the second set of amino acids are either DSG orDTG, where the last amino acid of the second set of special amino acidsis the last special amino acid, G, which is operably linked to anynumber of amino acids from 50 to 170 amino acids, which may be any aminoacids. In preferred embodiments, the last special amino acid is operablylinked to a peptide of about 100 to 170 amino acids or about 142-163amino acids. In particular embodiments, the last special amino acid isoperably linked to a peptide of about 142 amino acids, or about 163amino acids, or about 170 amino acids. For example, the polypeptidecomprises a sequence that is at least 95% identical (and preferably 100%identical) to an aspartyl protease sequence as described herein. In oneparticular embodiment, the second set of special amino acids iscomprised of the peptide with the amino acid sequence DSG. Optionally,the amino acid polypeptide is operably linked to a peptide purificationtag, such as purification tag which is six histidine. In one variation,the first set of special amino acids are on one polypeptide and thesecond set of special amino acids are on a second polypeptide, whereboth first and second polypeptide have at lease 50 amino acids, whichmay be any amino acids. In one embodiment of this type, both of thepolypeptides are in the same vessel. The invention further includes aprocess of making any of the polynucleotides, vectors, or cellsdescribed herein; and a process of making any of the polypeptidesdescribed herein.

In yet another related aspect, the invention provides a purifiedpolynucleotide comprising a nucleotide sequence that encodes apolypeptide having aspartyl protease activity, wherein the polypeptidehas an amino acid sequence characterized by: (a) a first tripeptidesequence DTG; (b) a second tripeptide sequence selected from the groupconsisting of DSG and DTG; and (c) about 100 to 300 amino acidsseparating the first and second tripeptide sequences, wherein thepolypeptide cleaves the beta secretase cleavage site of amyloid proteinprecursor. In one embodiment, the polypeptide comprises an amino acidsequence depicted in SEQ ID NO: 2 or 4, whereas in another embodiment,the polypeptide comprises an amino acid sequence other than the aminoacid sequences set forth in SEQ ID NOs: 2 and 4. Similarly, theinvention provides a purified polynucleotide comprising a nucleotidesequence that encodes a polypeptide that cleaves the beta secretasecleavage site of amyloid protein precursor; wherein the polynucleotideincludes a strand that hybridizes to one or more of SEQ ID NOs: 3, 5,and 7 under the following hybridization conditions: hybridizationovernight at 42° C. for 2.5 hours in 6×SSC/0.1% SDS, followed by washingin 1.0×SSC at 65° C., 0.1% SDS. In one embodiment, the polypeptidecomprises an amino acid sequence depicted in SEQ ID NO: 2 or 4, whereasin another embodiment, the polypeptide comprises an amino acid sequenceother than the amino acid sequences set forth in SEQ ID NOs: 2 and 4.Likewise, the invention provides a purified polypeptide having aspartylprotease activity, wherein the polypeptide is encoded by polynucleotidesas described in the preceding sentences. The invention also provides avector or host cell comprising such polynucleotides, and a method ofmaking the polypeptides using the vectors or host cells to recombinantlyexpress the polypeptide.

In yet another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide, said polynucleotide encoding aHu-Asp polypeptide and having a nucleotide sequence at least 95%identical to a sequence selected from the group consisting of:

(a) a nucleotide sequence encoding a Hu-Asp polypeptide selected fromthe group consisting of Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b), whereinsaid Hu-Asp1, Hu-Asp2(a) and Hu-Asp2(b) polypeptides have the completeamino acid sequence of SEQ ID NO. 2, SEQ ID NO. 4, and SEQ ID NO. 6,respectively; and

(b) a nucleotide sequence complementary to the nucleotide sequence of(a).

Several species are particularly contemplated. For example, theinvention provides a nucleic acid molecule wherein said Hu-Asppolypeptide is Hu-Asp1, and said polynucleotide molecule of (a)comprises the nucleotide sequence of SEQ ID NO.1; and a nucleic acidmolecule wherein said Hu-Asp polypeptide is Hu-Asp2(a), and saidpolynucleotide molecule of (a) comprises the nucleotide sequence of SEQID NO. 3; and a nucleic acid molecule wherein said Hu-Asp polypeptide isHu-Asp2(b), and said polynucleotide molecule of (a) comprises thenucleotide sequence of SEQ ID NO. 5. In addition to the foregoing, theinvention provides an isolated nucleic acid molecule comprising apolynucleotide which hybridizes under stringent conditions to apolynucleotide having the nucleotide sequence in (a) or (b) as describedabove.

Additionally, the invention provides a vector comprising a nucleic acidmolecule as described in the preceding paragraph. In a preferredembodiment, the nucleic acid molecule is operably linked to a promoterfor the expression of a Hu-Asp polypeptide. Individual vectors whichencode Hu-Asp1, and Hu-Asp2(a), and Hu-Asp2(b) are all contemplated.Likewise, the invention contemplates a host cell comprising any of theforegoing vectors, as well as a method of obtaining a Hu-Asp polypeptidecomprising culturing such a host cell and isolating the Hu-Asppolypeptide. Host cells of the invention include bacterial cells, suchas E. coli, and eukaryotic cells. Among the eukaryotic cells that arecontemplated are insect cells, such as sf9 or High 5 cells; andmammalian cells, such as human, rodent, lagomorph, and primate.Preferred human cells include HEK293, and IMR-32 cells. Other preferredmammalian cells include COS-7, CHO-K1, Neuro-2A, and 3T3 cells. Alsoamong the eukaryotic cells that are contemplated are a yeast cell and anavian cell.

In a related aspect, the invention provides an isolated Hu-Asp1polypeptide comprising an amino acid sequence at least 95% identical toa sequence comprising the amino acid sequence of SEQ ID NO. 2. Theinvention also provides an isolated Hu-Asp2(a) polypeptide comprising anamino acid sequence at least 95% identical to a sequence comprising theamino acid sequence of SEQ ID NO. 4. The invention also provides anisolated Hu-Asp2(a) polypeptide comprising an amino acid sequence atleast 95% identical to a sequence comprising the amino acid sequence ofSEQ ID NO. 8.

In still another aspect, the invention provides an isolated antibodythat binds specifically to any Hu-Asp polypeptide described herein,especially the polypeptide described in the preceding paragraphs.

The invention also provides several assays involving aspartyl proteaseenzymes of the invention. For example, the invention provides

a method to identify a cell that can be used to screen for inhibitors ofβ secretase activity comprising:

(a) identifying a cell that expresses a protease capable of cleaving APPat the β secretase site, comprising:

i) collect the cells or the supernatant from the cells to be identified

ii) measure the production of a critical peptide, where the criticalpeptide is selected from the group consisting of either the APPC-terminal peptide or soluble APP,

iii) select the cells which produce the critical peptide.

In one variation, the cells are collected and the critical peptide isthe APP C-terminal peptide created as a result of the β secretasecleavage. In another variation, the supernatant is collected and thecritical peptide is soluble APP, where the soluble APP has a C-terminuscreated by β secretase cleavage. In preferred embodiments, the cellscontain any of the nucleic acids or polypeptides described above and thecells are shown to cleave the β secretase site of any peptide having thefollowing peptide structure, P2, P1, P1′, P2′ (SEQ ID NO: 72), where P2is K or N, where P1 is M or L, where P1′ is D, where P2′ is A. In oneembodiment P2 is K and P1 is M and in another embodiment P2 is N and P1is L.

In still another aspect, the invention provides novel isoforms ofamyloid protein precursor (APP) where the last two carboxy terminusamino acids of that isoform are both lysine residues. In this context,the term “ isoform” is defined as any APP polypeptide, including APPvariants (including mutations), and APP fragments that exists in humans,such as those described in U.S. Pat. No. 5,766,846, col 7, lines 45-67,incorporated into this document by reference, modified as describedherein by the inclusion of two C-terminal lysine residues. For example,the invention provides a polypeptide comprising the isoform known asAPP695, modified to include two lysine residues as its last two carboxyterminus amino acids. An exemplary polypeptide comprises the amino acidsequence set forth in SEQ ID NO. 16. The invention further includes APPisoform variants as set forth in SEQ ID NOs. 18 and 20. The inventionfurther includes all polynucleotides that encode an APP protein that hasbeen modified to include two C-terminal lysines; as well has anyeukaryotic cell line comprising such nucleic acids or polypeptides.Preferred cell lines include a mammalian cell line (e.g., HEK293,Neuro2a).

Thus, in one embodiment, the invention provides a polypeptide comprisingthe amino acid sequence of a mammalian amyloid protein precursor (APP)or fragment thereof containing an APP cleavage site recognizable by amammalian β-secretase, and further comprising two lysine residues at thecarboxyl terminus of the amino acid sequence of the mammalian APP or APPfragment. As taught herein in detail, the addition of two additionallysine residues to APP sequences has been found to greatly increase Aβprocessing of the APP in APP processing assays. Thus, the di-lysinemodified APP reagents of the invention are particularly useful in assaysto identify modulators of Aβ production, for use in designingtherapeutics for the treatment or prevention of Alzheimer's disease. Inone embodiment, the polypeptide comprises the complete amino acidsequence of a mammalian amyloid protein precursor (APP), and furthercomprises the two lysine residues at the carboxyl terminus of the aminoacid sequence of the mammalian amyloid protein precursor. In analternative embodiment, the polypeptide comprises only a fragment of theAPP, the fragment containing at least that portion of APP that iscleaved by a mammalian β-secretase in the formation of Aβ peptides.

The practice of assays that monitor cleavage of APP can be facilitatedby attaching a marker to a portion of the APP. Measurement of retainedor liberated marker can be used to quantitate the amount of APP cleavagethat occurs in the assay, e.g., in the presence or absence of a putativemodulator of cleavage activity. Thus, in one preferred embodiment, thepolypeptide of the invention further includes a marker. For example, themarker comprises a reporter protein amino acid sequence attached to theAPP amino acid sequence. Exemplary reporter proteins include afluorescing protein (e.g., green fluorescing proteins, luciferase) or anenzyme that is used to cleave a substrate to produce a colorimetriccleavage product. Also contemplated are tag sequences which are commonlyused as epitopes for quantitative immunoassays.

In a preferred embodiment, the di-lysine-modified APP of the inventionis a human APP. For example, human APP isoforms such as APP695, APP751,and APP770, modified to include the two lysines, are contemplated. In apreferred embodiment, the APP isoform comprises at least one variationselected from the group consisting of a Swedish KM→NL mutation and aLondon V717-F mutation, or any other mutation that has been observed ina subpopulation that is particularly prone to development of Alzheimer'sdisease. These mutations are recognized as mutations that influence APPprocessing into Aβ. In a highly preferred embodiment, the APP protein orfragment thereof comprises the APP-Sw β-secretase peptide sequence NLDA,(SEQ ID. NO:66) which is associated with increased levels of Aβprocessing and therefore is particularly useful in assays relating toAlzheimer's research. More particularly, the APP protein or fragmentthereof preferably comprises the APP-Sw β-secretase peptide sequenceSEVNLDAEFR (SEQ ID NO: 63).

In one preferred embodiment, the APP protein or fragment thereof furtherincludes an APP transmembrane domain carboxy-terminal to the APP-Swβ-secretase peptide sequence. Polypeptides that include the TM domainare particularly useful in cell-based APP processing assays. Incontrast, embodiments lacking the TM domain are useful in cell-freeassays of APP processing.

In addition to working with APP from humans and various animal models,researchers in the field of Alzheimer's also have construct chimeric APPpolypeptides which include stretches of amino acids from APP of onespecies (e.g., humans) fused to streches of APP from one or more otherspecies (e.g., rodent). Thus, in another embodiment of the polypeptideof the invention, the APP protein or fragment thereof comprises achimeric APP, the chimeric APP including partial APP amino acidsequences from at least two species. A chimeric APP that includes aminoacid sequence of a human APP and a rodent APP is particularlycontemplated.

In a related aspect, the invention provides a polynucleotide comprisinga nucleotide sequence that encodes a polypeptide as described in thepreceding paragraphs. Such a polynucleotide is useful for recominantexpression of the polypeptide of the invention for use in APP processingassays. In addition, the polynucleotide is useful for transforming intocells to produce recombinant cells that express the polypeptide of theinvention, which cells are useful in cell-based assays to identifymodulators of APP processing. Thus, in addition to polynucleotides, theinvention provides a vector comprising such polynucleotides, especiallyexpression vectors where the polynucleotide is operably linked to apromoter to promote expression of the polypeptide encoded by thepolynucleotide in a host cell. The invention further provides a hostcell transformed or transfected with a polynucleotide according to claim14 or a vector according to claim 15 or 16. Among the preferred hostcells are mammalian cells, especially human cells.

In another, related embodiment, the invention provides a polypeptideuseful for assaying for modulators of β-secretase activity, saidpolypeptide comprising an amino acid sequence of the formulaNH₂—X—Y—Z—KK—COOH; wherein X, Y, and Z each comprise an amino acidsequence of at least one amino acid; wherein-NH₂—X comprises anamino-terminal amino acid sequence having at least one amino acidresidue; wherein Y comprises an amino acid sequence of a β-secretaserecognition site of a mammalian amyloid protein precursor (APP); andwherein Z—KK—COOH comprises a carboxy-terminal amino acid sequenceending in two lysine (K) residues. In one preferred variation, thecarboxyl-terminal amino acid sequence Z includes a hyrdrophobic domainthat is a transmembrane domain in host cells that express thepolypeptide. Host cells that express such a polypeptide are particularlyuseful in assays described herein for identifying modulators of APPprocessing. In another preferred variation, the amino-terminal aminoacid sequence X includes an amino acid sequence of a reporter or markerprotein, as described above. In still another preferred variation, theβ-secretase recognition site Y comprises the human APP-Sw β-secretasepeptide sequence NLDA (SEQ ID NO:66). It will be apparent that thesepreferred variations are not mutually exclusive of each other—they maybe combined in a single polypeptide. The invention further provides apolynucleotide comprising a nucleotide sequence that encodes suchpolypeptides, vectors which comprise such polynucleotides, and hostcells which comprises such vectors, polynucleotides, and/orpolypeptides.

In yet another aspect, the invention provides a method for identifyinginhibitors of an enzyme that cleaves the beta secretase cleavable siteof APP comprising:

a) culturing cells in a culture medium under conditions in which theenzyme causes processing of APP and release of amyloid beta-peptide intothe medium and causes the accumulation of CTF99 fragments of APP in celllysates,

b) exposing the cultured cells to a test compound; and specificallydetermining whether the test compound inhibits the function of theenzyme by measuring the amount of amyloid beta-peptide released into themedium and/or the amount of CTF99 fragments of APP in cell lysates;

c) identifying test compounds diminishing the amount of soluble amyloidbeta peptide present in the culture medium and diminution of CTF99fragments of APP in cell lysates as Asp2 inhibitors. In preferredembodiments, the cultured cells are a human, rodent or insect cell line.It is also preferred that the human or rodent cell line exhibits βsecretase activity in which processing of APP occurs with release ofamyloid beta-peptide into the culture medium and accumulation of CTF99in cell lysates. Among the contemplated test compounds are antisenseoligomers directed against the enzyme that exhibits β secretaseactivity, which oligomers reduce release of soluble amyloid beta-peptideinto the culture medium and accumulation of CTF99 in cell lysates.

In yet another aspect, the invention provides a method for theidentification of an agent that decreases the activity of a Hu-Asppolypeptide selected from the group consisting of Hu-Asp1, Hu-Asp2(a),and Hu-Asp2(b), the method comprising:

a) determining the activity of said Hu-Asp polypeptide in the presenceof a test agent and in the absence of a test agent; and

b) comparing the activity of said Hu-Asp polypeptide determined in thepresence of said test agent to the activity of said Hu-Asp polypeptidedetermined in the absence of said test agent; whereby a lower level ofactivity in the presence of said test agent than in the absence of saidtest agent indicates that said test agent has decreased the activity ofsaid Hu-Asp polypeptide.

In a related aspect, the invention provides a method for assaying formodulators of β-secretase activity, comprising the steps of:

(a) contacting a first composition with a second composition both in thepresence and in the absence of a putative modulator compound, whereinthe first composition comprises a mammalian β-secretase polypeptide orbiologically active fragment thereof, and wherein the second compositioncomprises a substrate polypeptide having an amino acid sequencecomprising a β-secretase cleavage site; (b) measuring cleavage of thesubstrate polypeptide in the presence and in the absence of the putativemodulator compound; and (c) identifying modulators of β-secretaseactivity from a difference in cleavage in the presence versus in theabsence of the putative modulator compound. A modulator that is aβ-secretase antagonist (inhibitor) reduces such cleavage, whereas amodulator that is a β-secretase agonist increases such cleavage. Sincesuch assays are relevant to development of Alzheimer's diseasetherapeutics for humans, it will be readily apparent that, in onepreferred embodiment, the first composition comprises a purified humanAsp2 polypeptide. In one variation, the first composition comprises asoluble fragment of a human Asp2 polypeptide that retains Asp2β-secretase activity. Several such fragments (including ΔTM fragments)are described herein in detail. Thus, in a particular embodiment, thesoluble fragment is a fragment lacking an Asp2 transmembrane domain.

The β-secretase cleavage site in APP is known, and it will beappreciated that the assays of the invention can be performed witheither intact APP or fragments or analogs of APP that retain theβ-secretase recognition and cleavage site. Thus, in one variation, thesubstrate polypeptide of the second composition comprises the amino acidsequence SEVNLDAEFR, (SEQ ID NO:63) which includes the β-secretaserecognition site of human APP that contains the “Swiss” mutation. Inanother variation, the substrate polypeptide of the second compositioncomprises the amino acid sequence EVKMDAEF (SEQ ID NO:67). In anothervariation, the second composition comprises a polypeptide having anamino acid sequence of a human amyloid precursor protein (APP). Forexample, the human amyloid precursor protein is selected from the groupconsisting of: APP695, APP751, and APP770. Preferably, the human amyloidprecursor protein (irrespective of isoform selected) includes at leaston mutation selected from a KM→NL Swiss mutation and a V→F Londonmutation. As explained elsewhere, one preferred embodiment involves avariation wherein the polypeptide having an amino acid sequence of ahuman APP further comprises an amino acid sequence comprising a markersequence attached amino-terminal to the amino acid sequence of the humanamyloid precursor protein. Preferably, the polypeptide having an aminoacid sequence of a human APP further comprises two lysine residuesattached to the carboxyl terminus of the amino acid sequence of thehuman APP. The assays can be performed in a cell free setting, usingcell-free enzyme and cell-free substrate, or can be performed in acell-based assay wherein the second composition comprises a eukaryoticcell that expresses amyloid precursor protein (APP) or a fragmentthereof containing a β-secretase cleavage site. Preferably, the APPexpressed by the host cell is an APP variant that includes twocarboxyl-terminal lysine residues. It will also be appreciated that theβ-secretase enzyme can be an enzyme that is expressed on the surface ofthe same cells.

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide that codes for a polypeptide selected fromthe group consisting of human aspartyl proteases. In particular, humanaspartyl protease 1 (Hu-Asp1) and two alternative splice variants ofhuman aspartyl protease-2 (Hu-Asp2), a “long” (L) form designated hereinas Hu-Asp2(a) and a “short” (S) form designated Hu-Asp2(b). As usedherein, all references to “Hu-Asp” should be understood to refer to allof Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b). In addition, as used herein, allreferences to “Hu-Asp2” should be understood to refer to both Hu-Asp2(a)and Hu-Asp2(b). Hu-Asp1 is expressed most abundantly in pancreas andprostate tissues, while Hu-Asp2(a) and Hu-Asp2(b) are expressed mostabundantly in pancreas and brain tissues. The invention also providesisolated Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b) polypeptides, as well asfragments thereof which exhibit aspartyl protease activity.

In a preferred embodiment, the nucleic acid molecules comprise apolynucleotide having a nucleotide sequence selected from the groupconsisting of residues 1-1554 of SEQ ID NO. 1, encoding Hu-Asp1,residues 1-1503 of SEQ ID NO. 3, encoding Hu-Asp2(a), and residues1-1428 of SEQ ID NO.5, encoding Hu-Asp2(b). In another aspect, theinvention provides an isolated nucleic acid molecule comprising apolynucleotide which hybridizes under stringent conditions to apolynucleotide encoding Hu-Asp1, Hu-Asp2(a), Hu-Asp-2(b), or fragmentsthereof European patent application EP 0 848 062 discloses a polypeptidereferred to as “Asp 1,” that bears substantial homology to Hu-Asp1,while international application WO 98/22597 discloses a polypeptidereferred to as “Asp 2,” that bears substantial homology to Hu-Asp2(a).

The present invention also provides vectors comprising the isolatednucleic acid molecules of the invention, host cells into which suchvectors have been introduced, and recombinant methods of obtaining aHu-Asp1, Hu-Asp2(a), or Hu-Asp2(b) polypeptide comprising culturing theabove-described host cell and isolating the relevant polypeptide.

In another aspect, the invention provides isolated Hu-Asp1, Hu-Asp2(a),and Hu-Asp2(b) polypeptides, as well as fragments thereof. In apreferred embodiment, the Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b)polypeptides have the amino acid sequence given in SEQ ID NO. 2, SEQ IDNO. 4, or SEQ ID NO.6, respectively. The present invention alsodescribes active forms of Hu-Asp2, methods for preparing such activeforms, methods for preparing soluble forms, methods for measuringHu-Asp2 activity, and substrates for Hu-Asp2 cleavage. The inventionalso describes antisense oligomers targeting the Hu-Asp1, Hu-Asp2(a) andHu-Asp2(b) mRNA transcripts and the use of such antisense reagents todecrease such mRNA and consequently the production of the correspondingpolypeptide. Isolated antibodies, both polyclonal and monoclonal, thatbinds specifically to any of the Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b)polypeptides of the invention are also provided.

The invention also provides a method for the identification of an agentthat modulates the activity of any of Hu-Asp-1, Hu-Asp2(a), andHu-Asp2(b). The inventions describes methods to test such agents incell-free assays to which Hu-Asp2 polypeptide is added, as well asmethods to test such agents in human or other mammalian cells in whichHu-Asp2 is present.

Additional features and variations of the invention win be apparent tothose skilled in the art from the entirety of this application,including the drawing and detailed description, and all such featuresare intended as aspects of the invention. Likewise, features of theinvention described herein can be recombined into additional embodimentsthat are also intended as aspects of the invention, irrespective ofwhether the combination of features is specifically mentioned above asan aspect or embodiment of the invention. Also, only such limitationswhich are described herein as critical to the invention should be viewedas such; variations of the invention lacking limitations which have notbeen described herein as critical are intended as aspects of theinvention.

In addition to the foregoing, the invention includes, as an additionalaspect, all embodiments of the invention narrower in scope in any waythan the variations specifically mentioned above. Although theapplicant(s) invented the full scope of the claims appended hereto, theclaims appended hereto are not intended to encompass within their scopethe prior art work of others. Therefore, in the event that statutoryprior art within the scope of a claim is brought to the attention of theapplicants by a Patent Office or other entity or individual, theapplicant(s) reserve the right to exercise amendment rights underapplicable patent laws to redefine the subject matter of such a claim tospecifically exclude such statutory prior art or obvious variations ofstatutory prior art from the scope of such a claim. Variations of theinvention defined by such amended claims also are intended as aspects ofthe invention.

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

Sequence ID No. 1: Human Asp-1, nucleotide sequence.

Sequence ID No. 3: Human Asp-2(a), nucleotide sequence.

Sequence ID No. 4: Human Asp-2(a), predicted amino acid sequence. TheAsp2(a) amino acid sequence includes a putative signal peptidecomprising residues 1 to 21; and a putative pre-propeptide after thesignal peptide that extends through residue 45 (as assessed byprocessing observed of recombinant Asp2(a) in CHO cells), and a putativepropeptide that may extend to at least about residue 57, based on theobservation of an observed GRR↓GS (SEQ ID NO:68) sequence which hascharacteristics of a protease recognition sequence. The Asp2(a) furtherincludes a transmembrane domain comprising residues 455-477, acytoplasmic domain comprising residues 478-501, and a putativealpha-helical spacer region, comprising residues 420-454, believed to beunnecessary for proteolytic activity, between the protease catalyticdomain and the transmembrane domain.

Sequence ID No. 5: Human Asp-2(b), nucleotide sequence.

Sequence ID No. 6: Human Asp-2(b), predicted amino acid sequence. TheAsp2(b) amino acid sequence includes a putative signal peptide,pre-propeptide, and propeptide as described above for Asp2(a). TheAsp2(b) further includes a transmembrane domain comprising residues430-452, a cytoplasmic domain comprising residues 453-476, and aputative alpha-helical spacer region, comprising residues 395-429,believed to be unnecessary for proteolytic activity, between theprotease catalytic domain and the transmembrane domain.

Sequence ID No. 7: Murine Asp-2(a), nucleotide sequence.

Sequence ID No. 8: Murine Asp-2(a), predicted amino acid sequence. Theproteolytic processing of murine Asp2(a) is believed to be analogous tothe processing described above for human Asp2(a). In addition, a variantlacking amino acid residues 190-214 of SEQ ID No: 8 is specificallycontemplated as a murine Asp2(b) polypeptide (SEQ ID NO: 73).

Sequence ID No. 9: Human APP695, nucleotide sequence.

Sequence ID No.10: Human APP695, predicted amino acid sequence.

Sequence ID No.11: Human APP695-Sw, nucleotide sequence.

Sequence ID No.12: Human APP695-Sw, predicted amino acid sequence. Inthe APP695 isoform, the Sw mutation is characterized by a KM→NLalteration at positions 595-596 (compared to normal APP695).

Sequence ID No.13: Human APP695-VF, nucleotide sequence.

Sequence ID No.14: Human APP695-VF, predicted amino acid sequence. Inthe APP 695 isoform, the VF mutation is characterized by a V→Falteration at position 642 (compared to normal APP 695).

Sequence ID No.15: Human APP695-KK, nucleotide sequence.

Sequence ID No.16: Human APP695-KK, predicted amino acid sequence.(APP695 with two carboxy-terminal lysine residues.)

Sequence ID No.17: Human APP695-Sw-KK, nucleotide sequence.

Sequence ID No.18: Human APP695-Sw-KK, predicted amino acid sequence.

Sequence ID No.19: Human APP695-VF-KK, nucleotide sequence.

Sequence ID No.20: Human APP695-VF-KK, predicted amino acid sequence.

Sequence ID No.21: T7-Human-pro-Asp-2(a)ΔTM, nucleotide sequence.

Sequence ID No.22: T7-Human-pro-Asp-2(a)ΔTM, amino acid sequence.

Sequence ID No.23: T7-Caspase-Human-pro-Asp-2(a)ΔTM, nucleotidesequence.

Sequence ID No.24: T7-Caspase-Human-pro-Asp-2(a)ΔTM amino acid sequence.

Sequence ID No.25: Human-pro-Asp-2(a)ΔTM (low GC), nucleotide sequence.

Sequence ID No.26: Human-pro-Asp- 2(a)ΔTM (low GC), amino acid sequence.

Sequence ID No.27: T7-Caspase-Caspase 8 cleavage-Human-pro-Asp-2(a)ΔTM,nucleotide sequence.

Sequence ID No.28: T7-Caspase-Caspase 8 cleavage-Human-pro-Asp-2(a)ΔTM,amino acid sequence.

Sequence ID No.29: Human Asp-2 (a)ΔTM, nucleotide sequence.

Sequence ID No.30: Human Asp-2(a)ΔTM amino acid sequence.

Sequence ID No.31: Human Asp-2(a)ΔTM(His)₆, nucleotide sequence.

Sequence ID No. 32: Human Asp-2(a)ΔTM(His)₆, amino acid sequence.

Sequence ID Nos. 33-49 are short synthetic peptide and oligonucleotidesequences that are described below in the Detailed Description of theInvention.

Sequence ID No. 50: Human Asp2(b)ΔTM polynucleotide sequence.

Sequence ID No. 51: Human Asp2(b)ΔTM polypeptide sequence (exemplaryvariant of Human Asp2(b) lacking transmembrane and intracellular domainsof Hu-Asp2(b) set forth in SEQ ID NO: 6.

Sequence ID No. 52: Human Asp2(b)ΔTM(His)₆ polynucleotide sequence.

Sequence ID No. 53: Human Asp2(b)ΔTM(His)₆ polypeptide sequence (HumanAsp2(b)ΔTM with six histidine tag attached to C-terminus).

Sequence ID No. 54: Human APP770-encoding polynucleotide sequence.

Sequence ID No. 55: Human APP770 polypeptide sequence. To introduce theKM→NL Swedish mutation, residues KM at positions 670-71 are changed toNL. To introduce the V→F London mutation, the V residue at position 717is changed to F.

Sequence ID No. 56: Human APP751 encoding polynucleotide sequence.

Sequence ID No. 57: Human APP751 polypeptide sequence (Human APP751isoform).

Sequence ID No. 58: Human APP770-KK encoding polynucleotide sequence.

Sequence ID No. 59: Human APP770-KK polypeptide sequence. (Human APP770isoform to which two C-terminal lysines have been added).

Sequence ID No. 60: Human APP751-KK encoding polynucleotide sequence.

Sequence ID No. 61: Human APP751-KK polypeptide sequence (Human APP751isoform to which two C-terminal lysines have been added).

Sequence ID No. 62-65: Various short peptide sequences described indetail in detailed description.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FIG. 1 shows the nucleotide (SEQ ID NO:1) and predicted aminoacid sequence (SEQ ID NO:2) of human Asp1.

FIG. 2: FIG. 2 shows the nucleotide (SEQ ID NO: 5) and predicted aminoacid sequence (SEQ ID NO: 6) of human Asp2(b).

FIG. 3: FIG. 3 shows the nucleotide (SEQ ID NO: 3) and predicted aminoacid sequence (SEQ ID NO: 4) of human Asp2(a).

FIG. 4: FIG. 4 shows the nucleotide (SEQ ID No. 7) and predicted aminoacid sequence (SEQ ID No. 8) of murine Asp2(a).

FIG. 5: FIG. 5 shows the BestFit alignment of the predicted amino acidsequences of Hu-Asp2(a) (SEQ ID. NO:4) and murine Asp2(a) (SEQ ID No:8).

FIG. 6: FIG. 6 shows the nucleotide (SEQ ID No. 21) and predicted aminoacid sequence (SEQ ID No. 22) of T7-Human-pro-Asp-2(a)ΔTM.

FIG. 7: FIG. 7 shows the nucleotide (SEQ ID No. 23) and predicted aminoacid sequence (SEQ ID No. 24) of T7-caspase-Human-pro-Asp-2(a)ΔTM.

FIG. 8: FIG. 8 shows the nucleotide (SEQ ID No. 25) and predicted aminoacid sequence (SEQ ID No. 26) of Human-pro-Asp-2(a)ΔTM (low GC)

FIG. 9: Western blot showing reduction of CTF99 production by HEK125.3cells transfected with antisense oligomers targeting the Hu-Asp2 mRNA.

FIG. 10: Western blot showing increase in CTF99 production in mouseNeuro-2a cells cotransfected with APP-KK with and without Hu-Asp2 onlyin those cells cotransfected with Hu-Asp2. A further increase in CTF99production is seen in cells cotransfected with APP-Sw-KK with andwithout Hu-Asp2 only in those cells cotransfected with Hu-Asp2.

FIG. 11: FIG. 11 shows the predicted amino acid sequence (SEQ ID No. 30)of Human-Asp2(a)ΔTM.

FIG. 12: FIG. 12 shows the predicted amino acid sequence (SEQ ID No. 30)of Human-Asp2(a)ΔTM(His)₆.

DETAILED DESCRIPTION OF THE INVENTION

A few definitions used in this invention follow, most definitions to beused are those that would be used by one ordinarily skilled in the art.

The term “β amyloid peptide” means any peptide resulting from betasecretase cleavage of APP. This includes peptides of 39, 40, 41, 42 and43 amino acids, extending from the β-secretase cleavage site to 39, 40,41, 42 and 43 amino acids C-terminal to the β-secretase cleavage site. βamyloid peptide also includes sequences 1-6, SEQ ID NOs. 1-6 of U.S.Pat. No. 5,750,349, issued May 12, 1998 (incorporated into this documentby reference). A β-secretase cleavage fragment disclosed here is calledCTF-99, which extends from β-secretase cleavage site to the carboxyterminus of APP.

When an isoform of APP is discussed then what is meant is any APPpolypeptide, including APP variants (including mutations), and APPfragments that exists in humans such as those described in U.S. Pat. No.5,766,846, col 7, lines 45-67, incorporated into this document byreference.

The term “β-amyloid precursor protein” (APP) as used herein is definedas a polypeptide that is encoded by a gene of the same name localized inhumans on the long arm of chromosome 21 and that includes “βAP—here“β-amyloid protein” see above, within its carboxyl third. APP is aglycosylated, single-membrane spanning protein expressed in a widevariety of cells in many mammalian tissues. Examples of specificisotypes of APP which are currently known to exist in humans are the 695amino acid polypeptide described by Kang et. al. (1987) Nature325:733-736 which is designated as the “normal” APP (SEQ ID NOs: 9-10);the 751 amino acid polypeptide described by Ponte et al. (1988) Nature331:525-527 (1988) and Tanzi et al. (1988) Nature 331:528-530 (SEQ IDNOs: 56-57); and the 770-amino acid polypeptide described by Kitaguchiet. al. (1988) Nature 331:530-532 (SEQ ID NOs: 54-55). Examples ofspecific variants of APP include point mutation which can differ in bothposition and phenotype (for review of known variant mutation see Hardy(1992) Nature Genet. 1:233-234). All references cited here incorporatedby reference. The term “APP fragments” as used herein refers tofragments of APP other than those which consist solely of βAP or βAPfragments. That is, APP fragments will include amino acid sequences ofAPP in addition to those which form intact βAP or a fragment of βAP.

When the term “any amino acid” is used, the amino acids referred to areto be selected from the following, three letter and single letterabbreviations—which may also be used, are provided as follows:

Alanine, Ala, A; Arginine, Arg, R; Asparagine, Asn, N; Aspartic acid,Asp, D; Cysteine, Cys, C; Glutamine, Gln, Q; Glutamic Acid, Glu, E;Glycine, Gly, G; Histidine, His, H; Isoleucine, Ile, I; Leucine, Leu, L;Lysine, Lys, K; Methionine, Met, M; Phenylalanine, Phe, F; Proline, Pro,P; Serine, Ser, S; Threonine, Thr, T; Tryptophan, Trp, W; Tyrosine, Tyr,Y; Valine, Val, V; Aspartic acid or Asparagine, Asx, B; Glutamic acid orGlutamine, Glx, Z; Any amino acid, Xaa, X.

The present invention describes a method to scan gene databases for thesimple active site motif characteristic of aspartyl proteases.Eukaryotic aspartyl proteases such as pepsin and renin possess atwo-domain structure which folds to bring two aspartyl residues intoproximity within the active site. These are embedded in the shorttripeptide motif DTG, or more rarely, DSG. Most aspartyl proteases occuras proenzyme whose N-terminus must be cleaved for activation. The DTG orDSG active site motif appears at about residue 65-70 in the proenzyme(prorenin, pepsinogen), but at about residue 25-30 in the active enzymeafter cleavage of the N-terminal prodomain. The limited length of theactive site motif makes it difficult to search collections of short,expressed sequence tags (EST) for novel aspartyl proteases. ESTsequences typically average 250 nucleotides or less, and so would encode80-90 amino acid residues or less. That would be too short a sequence tospan the two active site motifs. The preferred method is to scandatabases of hypothetical or assembled protein coding sequences. Thepresent invention describes a computer method to identify candidateaspartyl proteases in protein sequence databases. The method was used toidentify seven candidate aspartyl protease sequences in theCaenorhabditis elegans genome. These sequences were then used toidentify by homology search Hu-Asp1 and two alternative splice variantsof Hu-Asp2, designated herein as Hu-Asp2(a) and Hu-Asp2(b).

In a major aspect of the invention disclosed here we provide newinformation about APP processing. Pathogeneic processing of the amyloidprecursor protein (APP) via the Aβ pathway requires the sequentialaction of two proteases referred to as β-secretase and γ-secretase.Cleavage of APP by the β-secretase and γ-secretase generates theN-terminus and C-terminus of the Aβ peptide, respectively. Because overproduction of the Aβ peptide, particularly the Aβ₁₋₄₂, has beenimplicated in the initiation of Alzheimer's disease, inhibitors ofeither the β-secretase and/or the γ-secretase have potential in thetreatment of Alzheimer's disease. Despite the importance of theβ-secretase and γ-secretase in the pathogenic processing of APP,molecular definition of these enzymes has not been accomplished to date.That is, it was not known what enzymes were required for cleavage ateither the β-secretase or the γ-secretase cleavage site. The sitesthemselves were known because APP was known and the Aβ₁₋₄₂, peptide wasknown, see U.S. Pat. Nos. 5,766,846 and 5,837,672, (incorporated byreference, with the exception to reference to “soluble” peptides). Butwhat enzyme was involved in producing the Aβ₁₋₄₂, peptide was unknown.

Alignment of the amino acid sequences of Hu-Asp2 with other knownaspartyl proteases reveals a similar domain organization. All of thesequences contain a signal sequence followed by a pro-segment and thecatalytic domain containing 2 copies of the aspartyl protease activesite motif (DTG/DSG) separated by approximately 180 amino acid residues.Comparison of the processing site for proteolytic removal of thepro-segment in the mature forms of pepsin A, pepsin C, cathepsin D,cathepsin E and renin reveals that the mature forms of these enzymescontain between 31-35 amino acid residues upstream of the first DTGmotif Inspection of this region in the Hu-Asp-2 amino acid sequenceindicates a preferred processing site within the sequence GRR↓GS (SEQ IDNO:68) as proteolytic processing of pro-protein precursors commonlyoccurs at site following dibasic amino acid pairs (eg. RR). Also,processing at this site would yield a mature enzyme with 35 amino acidresidues upstream of the first DTG, consistent with the processing sitesfor other aspartyl proteases. In the absence of self-activation ofHu-Asp2 or a knowledge of the endogenous protease that processes Hu-Asp2at this site, a recombinant form was engineered by introducing arecognition site for the PreSission protease (LEVLFQ↓GP (SEQ ID No:62))into the expression plasmids for bacterial, insect cell, and mammaliancell expression of pro-Hu-Asp2. In each case, the Gly residue in P1′position corresponds to the Gly residue 35 amino acids upstream of thefirst DTG motif in Hu-Asp2.

The present invention involves the molecular definition of several novelhuman aspartyl proteases and one of these, referred to as Hu-Asp-2(a)and Hu-Asp2(b), has been characterized in detail. Previous forms of asp1and asp 2 have been disclosed, see EP 0848062 A2 and EP 0855444A2,inventors David Powel et al., assigned to Smith Kline Beecham Corp.(incorporated by reference). Herein are disclosed old and new forms ofHu-Asp 2. For the first time they are expressed in active form, theirsubstrates are disclosed, and their specificity is disclosed. Prior tothis disclosure cell or cell extracts were required to cleave theβ-secretase site, now purified protein can be used in assays, alsodescribed here. Based on the results of (1) antisense knock outexperiments, (2) transient transfection knock in experiments, and (3)biochemical experiments using purified recombinant Hu-Asp-2, wedemonstrate that Hu-Asp-2 is the β-secretase involved in the processingof APP. Although the nucleotide and predicted amino acid sequence ofHu-Asp-2(a) has been reported, see above, see EP 0848062 A2 and EP0855444A2, no functional characterization of the enzyme was disclosed.Here the authors characterize the Hu-Asp-2 enzyme and are able toexplain why it is a critical and essential enzyme required in theformation of Aβ₁₋₄₂, peptide and possible a critical step in thedevelopment of AD.

In another embodiment the present invention also describes a novelsplice variant of Hu-Asp2, referred to as Hu-Asp-2(b), that has neverbefore been disclosed.

In another embodiment, the invention provides isolated nucleic acidmolecules comprising a polynucleotide encoding a polypeptide selectedfrom the group consisting of human aspartyl protease 1 (Hu-Asp1) and twoalternative splice variants of human aspartyl protease-2 (Hu-Asp2),designated herein as Hu-Asp2(a) and Hu-Asp2(b). As used herein, allreferences to “Hu-Asp2” should be understood to refer to both Hu-Asp2(a)and Hu-Asp2(b). Hu-Asp1 is expressed most abundantly in pancreas andprostate tissues, while Hu-Asp2(a) and Hu-Asp2(b) are expressed mostabundantly in pancreas and brain tissues. The invention also providesisolated Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b) polypeptides, as well asfragments thereof which exhibit aspartyl protease activity.

The predicted amino acid sequences of Hu-Asp1, Hu-Asp2(a) and Hu-Asp2(b)share significant homology with previously identified mammalian aspartylproteases such as pepsinogen A, pepsinogen B, cathepsin D, cathepsin E,and renin. P. B. Szecs, Scand J. Clin. Lab. Invest. 52:(Suppl. 210 5-22(1992)). These enzymes are characterized by the presence of a duplicatedDTG/DSG sequence motif. The Hu-Asp1 and HuAsp2 polypeptides disclosedherein also exhibit extremely high homology with the ProSite consensusmotif for aspartyl proteases extracted from the SwissProt database.

The nucleotide sequence given as residues 1-1554 of SEQ ID NO:1corresponds to the nucleotide sequence encoding Hu-Asp1, the nucleotidesequence given as residues 1-1503 of SEQ ID NO:3 corresponds to thenucleotide sequence encoding Hu-Asp2(a), and the nucleotide sequencegiven as residues 1-1428 of SEQ ID NO:5 corresponds to the nucleotidesequence encoding Hu-Asp2(b). The isolation and sequencing of DNAencoding Hu-Asp1, Hu-Asp2(a), and Hu-Asp2(b) is described below inExamples 1 and 2.

As is described in Examples 1 and 2, automated sequencing methods wereused to obtain the nucleotide sequence of Hu-Asp1, Hu-Asp2(a), andHu-Asp-2(b). The Hu-Asp nucleotide sequences of the present inventionwere obtained for both DNA strands, and are believed to be 100%accurate. However, as is known in the art, nucleotide sequence obtainedby such automated methods may contain some errors. Nucleotide sequencesdetermined by automation are typically at least about 90%, moretypically at least about 95% to at least about 99.9% identical to theactual nucleotide sequence of a given nucleic acid molecule. The actualsequence may be more precisely determined using manual sequencingmethods, which are well known in the art. An error in sequence whichresults in an insertion or deletion of one or more nucleotides mayresult in a frame shift in translation such that the predicted aminoacid sequence will differ from that which would be predicted from theactual nucleotide sequence of the nucleic acid molecule, starting at thepoint of the mutation. The Hu-Asp DNA of the present invention includescDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, andcombinations thereof Genomic Hu-Asp DNA may be obtained by screening agenomic library with the Hu-Asp2 cDNA described herein, using methodsthat are well known in the art, or with oligonucleotides chosen from theHu-Asp2 sequence that will prime the polymerase chain reaction (PCR).RNA transcribed from Hu-Asp DNA is also encompassed by the presentinvention.

Due to the degeneracy of the genetic code, two DNA sequences may differand yet encode identical amino acid sequences. The present inventionthus provides isolated nucleic acid molecules having a polynucleotidesequence encoding any of the Hu-Asp polypeptides of the invention,wherein said polynucleotide sequence encodes a Hu-Asp polypeptide havingthe complete amino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, or fragments thereof.

Also provided herein are purified Hu-Asp polypeptides, both recombinantand non-recombinant. Most importantly, methods to produce Hu-Asp2polypeptides in active form are provided. These include production ofHu-Asp2 polypeptides and variants thereof in bacterial cells, insectcells, and mammalian cells, also in forms that allow secretion of theHu-Asp2 polypeptide from bacterial, insect or mammalian cells into theculture medium, also methods to produce variants of Hu-Asp2 polypeptideincorporating amino acid tags that facilitate subsequent purification.In a preferred embodiment of the invention the Hu-Asp2 polypeptide isconverted to a proteolytically active form either in transformed cellsor after purification and cleavage by a second protease in a cell-freesystem, such active forms of the Hu-Asp2 polypeptide beginning with theN-terminal sequence TQHGIR (SEQ ID No:69) or ETDEEP (SEQ ID NO:70). Thesequence TQHGIR represents the amino-terminus of Asp2(a) or Asp2(b)beginning with residue 22 of SEQ ID NO: 4 or 6, after cleavage of aputative 21 residue signal peptide. Recombinant Asp2(a) expressed in andpurified from insect cells was observed to have this amino terminus,presumably as a result of cleavage by a signal peptidase. The sequenceETDEEP (SEQ ID NO:70) represents the amino-terminus of Asp2(a) orAsp2(b) beginning with residue 46 of SEQ ID NO: 4 or 6, as observed whenAsp2(a) has been recombinantly produced in CHO cells (presumably aftercleavage by both a rodent signal peptidase and another rodent peptidasethat removes a propeptide sequence). The Asp2(a) produced in the CHOcells possesses β-secretase activity, as described in greater detail inExamples 11 and 12. Variants and derivatives, including fragments, ofHu-Asp proteins having the native amino acid sequences given in SEQ IDNos: 2, 4, and 6 that retain any of the biological activities of Hu-Aspare also within the scope of the present invention. Of course, one ofordinary skill in the art will readily be able to determine whether avariant, derivative, or fragment of a Hu-Asp protein displays Hu-Aspactivity by subjecting the variant, derivative, or fragment to astandard aspartyl protease assay. Fragments of Hu-Asp within the scopeof this invention include those that contain the active site domaincontaining the amino acid sequence DTG, fragments that contain theactive site domain amino acid sequence DSG, fragments containing boththe DTG and DSG active site sequences, fragments in which the spacing ofthe DTG and DSG active site sequences has been lengthened, fragments inwhich the spacing has been shortened. Also within the scope of theinvention are fragments of Hu-Asp in which the transmembrane domain hasbeen removed to allow production of Hu-Asp2 in a soluble form. Inanother embodiment of the invention, the two halves of Hu-Asp2, eachcontaining a single active site DTG or DSG sequence can be producedindependently as recombinant polypeptides, then combined in solutionwhere they reconstitute an active protease.

Thus, the invention provides a purified polypeptide comprising afragment of a mammalian Asp2 protein, wherein said fragment lacks theAsp2 transmembrane domain of said Asp2 protein, and wherein thepolypeptide and the fragment retain the β-secretase activity of saidmammalian Asp2 protein. In a preferred embodiment, the purifiedpolypeptide comprises a fragment of a human Asp2 protein that retainsthe β-secretase activity of the human Asp2 protein from which it wasderived. Examples include:

a purified polypeptide that comprises a fragment of Asp2(a) having theamino acid sequence set forth in SEQ ID NO: 4, wherein the polypeptidelacks transmembrane domain amino acids 455 to 477 of SEQ ID NO: 4;

a purified polypeptide as described in the preceding paragraph thatfurther lacks cytoplasmic domain amino acids 478 to 501 of SEQ ID NO: 4;

a purified polypeptide as described in either of the precedingparagraphs that further lacks amino acids 420-454 of SEQ ID NO: 4, whichconstitute a putative alpha helical region between the catalytic domainand the transmembrane domain that is believed to be unnecessary forβ-secretase activity;

a purified polypeptide that comprises an amino acid sequence thatincludes amino acids 58 to 419 of SEQ ID NO: 4, and that lacks aminoacids 22 to 57 of SEQ ID NO: 4;

a purified polypeptide that comprises an amino acid sequence thatincludes amino acids 46 to 419 of SEQ ID NO: 4, and that lacks aminoacids 22 to 45 of SEQ ID NO: 4;

a purified polypeptide that comprises an amino acid sequence thatincludes amino acids 22 to 454 of SEQ ID NO: 4;

a purified polypeptide that comprises a fragment of Asp2(b) having theamino acid sequence set forth in SEQ ID NO: 6, and wherein saidpolypeptide lacks transmembrane domain amino acids 430 to 452 of SEQ IDNO: 6;

a purified polypeptide as described in the preceding paragraph thatfurther lacks cytoplasmic domain amino acids 453 to 476 of SEQ ID NO: 6;

a purified polypeptide as described in either of the preceding twoparagraphs that further lacks amino acids 395-429 of SEQ ID NO: 4, whichconstitute a putative alpha helical region between the catalytic domainand the transmembrane domain that is believed to be unnecessary forβ-secretase activity;

a purified polypeptide comprising an amino acid sequence that includesamino acids 58 to 394 of SEQ ID NO: 4, and that lacks amino acids 22 to57 of SEQ ID NO: 4;

a purified polypeptide comprising an amino acid sequence that includesamino acids 46 to 394 of SEQ ID NO: 4, and that lacks amino acids 22 to45 of SEQ ID NO: 4; and

a purified polypeptide comprising an amino acid sequence that includesamino acids 22 to 429 of SEQ ID NO: 4.

Also included as part of the invention is a purified polynucleotidecomprising a nucleotide sequence that encodes such polypeptides; avector comprising a polynucleotide that encodes such polypeptides; and ahost cell transformed or transfected with such a polynucleotide orvector.

Hu-Asp variants may be obtained by mutation of native Hu-Asp-encodingnucleotide sequences, for example. A Hu-Asp variant, as referred toherein, is a polypeptide substantially homologous to a native Hu-Asppolypeptide but which has an amino acid sequence different from that ofnative Hu-Asp because of one or more deletions, insertions, orsubstitutions in the amino acid sequence. The variant amino acid ornucleotide sequence is preferably at least about 80% identical, morepreferably at least about 90% identical, and most preferably at leastabout 95% identical, to a native Hu-Asp sequence. Thus, a variantnucleotide sequence which contains, for example, 5 point mutations forevery one hundred nucleotides, as compared to a native Hu-Asp gene, willbe 95% identical to the native protein. The percentage of sequenceidentity, also termed homology, between a native and a variant Hu-Aspsequence may also be determined, for example, by comparing the twosequences using any of the computer programs commonly employed for thispurpose, such as the Gap program (Wisconsin Sequence Analysis Package,Version 8 for Unix, Genetics Computer Group, University Research Park,Madison Wis.), which uses the algorithm of Smith and Waterman (Adv.Appl. Math. 2: 482-489 (1981)).

Alterations of the native amino acid sequence may be accomplished by anyof a number of known techniques. For example, mutations may beintroduced at particular locations by procedures well known to theskilled artisan, such as oligonucleotide-directed mutagenesis, which isdescribed by Walder et al. (Gene 42:133 (1986)); Bauer et al. (Gene37:73 (1985)); Craik (BioTechniques, January 1985, pp. 12-19); Smith etal. (Genetic Engineering: Principles and Methods, Plenum Press (1981));and U.S. Pat. Nos. 4,518,584 and 4,737,462.

Hu-Asp variants within the scope of the invention may compriseconservatively substituted sequences, meaning that one or more aminoacid residues of a Hu-Asp polypeptide are replaced by different residuesthat do not alter the secondary and/or tertiary structure of the Hu-Asppolypeptide. Such substitutions may include the replacement of an aminoacid by a residue having similar physicochemical properties, such assubstituting one aliphatic residue (Ile, Val, Leu or Ala) for another,or substitution between basic residues Lys and Arg, acidic residues Gluand Asp, amide residues Gln and Asn, hydroxyl residues Ser and Tyr, oraromatic residues Phe and Tyr. Further information regarding makingphenotypically silent amino acid exchanges may be found in Bowie et al.,Science 247:1306-1310 (1990). Other Hu-Asp variants which might retainsubstantially the biological activities of Hu-Asp are those where aminoacid substitutions have been made in areas outside functional regions ofthe protein.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringentconditions to a portion of the nucleic acid molecules described above,e.g., to at least about 15 nucleotides, preferably to at least about 20nucleotides, more preferably to at least about 30 nucleotides, and stillmore preferably to at least about from 30 to at least about 100nucleotides, of one of the previously described nucleic acid molecules.Such portions of nucleic acid molecules having the described lengthsrefer to, e.g., at least about 15 contiguous nucleotides of thereference nucleic acid molecule. By stringent hybridization conditionsis intended overnight incubation at about 42° C. for about 2.5 hours in6×SSC/0.1% SDS, followed by washing of the filters four times for 15minutes in 1.0×SSC at 65° C., 0.1% SDS.

Fragments of the Hu-Asp encoding nucleic acid molecules describedherein, as well as polynucleotides capable of hybridizing to suchnucleic acid molecules may be used as a probe or as primers in apolymerase chain reaction (PCR). Such probes may be used, e.g., todetect the presence of Hu-Asp nucleic acids in in vitro assays, as wellas in Southern and northern blots. Cell types expressing Hu-Asp may alsobe identified by the use of such probes. Such procedures are well known,and the skilled artisan will be able to choose a probe of a lengthsuitable to the particular application. For PCR, 5′ and 3′ primerscorresponding to the termini of a desired Hu-Asp nucleic acid moleculeare employed to isolate and amplify that sequence using conventionaltechniques.

Other useful fragments of the Hu-Asp nucleic acid molecules areantisense or sense oligonucleotides comprising a single stranded nucleicacid sequence capable of binding to a target Hu-Asp mRNA (using a sensestrand), or Hu-Asp DNA (using an antisense strand) sequence. In apreferred embodiment of the invention these Hu-Asp antisenseoligonucleotides reduce Hu-Asp MRNA and consequent production of Hu-Asppolypeptides.

In another aspect, the invention includes Hu-Asp polypeptides with orwithout associated native pattern glycosylation. Both Hu-Asp1 andHu-Asp2 have canonical acceptor sites for Asn-linked sugars, withHu-Asp1 having two of such sites, and Hu-Asp2 having four. Hu-Aspexpressed in yeast or mammalian expression systems (discussed below) maybe similar to or significantly different from a native Hu-Asppolypeptide in molecular weight and glycosylation pattern. Expression ofHu-Asp in bacterial expression systems will provide non-glycosylatedHu-Asp.

The polypeptides of the present invention are preferably provided in anisolated form, and preferably are substantially purified. Hu-Asppolypeptides may be recovered and purified from tissues, cultured cells,or recombinant cell cultures by well-known methods, including ammoniumsulfate or ethanol precipitation, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatography,lectin chromatography, and high performance liquid chromatography(HPLC). In a preferred embodiment, an amino acid tag is added to theHu-Asp polypeptide using genetic engineering techniques that are wellknown to practitioners of the art which include addition of sixhistidine amino acid residues to allow purification by binding to nickelimmobilized on a suitable support, epitopes for polyclonal or monoclonalantibodies including but not limited to the T7 epitope, the myc epitope,and the V5a epitope, and fusion of Hu-Asp2 to suitable protein partnersincluding but not limited to glutathione-S-transferase or maltosebinding protein. In a preferred embodiment these additional amino acidsequences are added to the C-terminus of Hu-Asp but may be added to theN-terminus or at intervening positions within the Hu-Asp2 polypeptide.

The present invention also relates to vectors comprising thepolynucleotide molecules of the invention, as well as host celltransformed with such vectors. Any of the polynucleotide molecules ofthe invention may be joined to a vector, which generally includes aselectable marker and an origin of replication, for propagation in ahost. Because the invention also provides Hu-Asp polypeptides expressedfrom the polynucleotide molecules described above, vectors for theexpression of Hu-Asp are preferred. The vectors include DNA encoding anyof the Hu-Asp polypeptides described above or below, operably linked tosuitable transcriptional or translational regulatory sequences, such asthose derived from a mammalian, microbial, viral, or insect gene.Examples of regulatory sequences include transcriptional promoters,operators, or enhancers, MRNA ribosomal binding sites, and appropriatesequences which control transcription and translation. Nucleotidesequences are operably linked when the regulatory sequence functionallyrelates to the DNA encoding Hu-Asp. Thus, a promoter nucleotide sequenceis operably linked to a Hu-Asp DNA sequence if the promoter nucleotidesequence directs the transcription of the Hu-Asp sequence.

Selection of suitable vectors to be used for the cloning ofpolynucleotide molecules encoding Hu-Asp, or for the expression ofHu-Asp polypeptides, will of course depend upon the host cell in whichthe vector will be transformed, and, where applicable, the host cellfrom which the Hu-Asp polypeptide is to be expressed. Suitable hostcells for expression of Hu-Asp polypeptides include prokaryotes, yeast,and higher eukaryotic cells, each of which is discussed below.

The Hu-Asp polypeptides to be expressed in such host cells may also befusion proteins which include regions from heterologous proteins. Suchregions may be included to allow, e.g., secretion, improved stability,or facilitated purification of the polypeptide. For example, a sequenceencoding an appropriate signal peptide can be incorporated intoexpression vectors. A DNA sequence for a signal peptide (secretoryleader) may be fused inframe to the Hu-Asp sequence so that Hu-Asp istranslated as a fusion protein comprising the signal peptide. A signalpeptide that is functional in the intended host cell promotesextracellular secretion of the Hu-Asp polypeptide. Preferably, thesignal sequence will be cleaved from the Hu-Asp polypeptide uponsecretion of Hu-Asp from the cell. Nonlimiting examples of signalsequences that can be used in practicing the invention include the yeastIfactor and the honeybee melatin leader in sf9 insect cells.

In a preferred embodiment, the Hu-Asp polypeptide will be a fusionprotein which includes a heterologous region used to facilitatepurification of the polypeptide. Many of the available peptides used forsuch a function allow selective binding of the fusion protein to abinding partner. For example, the Hu-Asp polypeptide may be modified tocomprise a peptide to form a fusion protein which specifically binds toa binding partner, or peptide tag. Nonlimiting examples of such peptidetags include the 6-His tag, thioredoxin tag, hemaglutinin tag, GST tag,and OmpA signal sequence tag. As will be understood by one of skill inthe art, the binding partner which recognizes and binds to the peptidemay be any molecule or compound including metal ions (e.g., metalaffinity columns), antibodies, or fragments thereof, and any protein orpeptide which binds the peptide, such as the FLAG tag.

Suitable host cells for expression of Hu-Asp polypeptides includesprokaryotes, yeast, and higher eukaryotic cells. Suitable prokaryotichosts to be used for the expression of Hu-Asp include bacteria of thegenera Escherichia, Bacillus, and Salmonella, as well as members of thegenera Pseudomonas, Streptomyces, and Staphylococcus. For expression in,e.g., E. coli, a Hu-Asp polypeptide may include an N-terminal methionineresidue to facilitate expression of the recombinant polypeptide in aprokaryotic host. The N-terminal Met may optionally then be cleaved fromthe expressed Hu-Asp polypeptide. Other N-terminal amino acid residuescan be added to the Hu-Asp polypeptide to facilitate expression inEscherichia coli including but not limited to the T7 leader sequence,the T7-caspase 8 leader sequence, as well as others leaders includingtags for purification such as the 6-His tag (Example 9). Hu-Asppolypeptides expressed in E. coli may be shortened by removal of thecytoplasmic tail, the transmembrane domain, or the membrane proximalregion. Hu-Asp polypeptides expressed in E. coli may be obtained ineither a soluble form or as an insoluble form which may or may not bepresent as an inclusion body. The insoluble polypeptide may be renderedsoluble by guanidine HCl, urea or other protein denaturants, thenrefolded into a soluble form before or after purification by dilution ordialysis into a suitable aqueous buffer. If the inactive proform of theHu-Asp was produced using recombinant methods, it may be rendered activeby cleaving off the prosegment with a second suitable protease such ashuman immunodeficiency virus protease.

Expression vectors for use in prokaryotic hosts generally comprises oneor more phenotypic selectable marker genes. Such genes generally encode,e.g., a protein that confers antibiotic resistance or that supplies anauxotrophic requirement. A wide variety of such vectors are readilyavailable from commercial sources. Examples include pSPORT vectors, pGEMvectors (Promega), pPROEX vectors (LTI, Bethesda, Md.), Bluescriptvectors (Stratagene), pET vectors (Novagen) and pQE vectors (Qiagen).

Hu-Asp may also be expressed in yeast host cells from genera includingSaccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S.cerevisiae and P. pastoris. Yeast vectors will often contain an originof replication sequence from a 2T yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Vectors replicable in both yeast and E. coli(termed shuttle vectors) may also be used. In addition to theabove-mentioned features of yeast vectors, a shuttle vector will alsoinclude sequences for replication and selection in E. coli. Directsecretion of Hu-Asp polypeptides expressed in yeast hosts may beaccomplished by the inclusion of nucleotide sequence encoding the yeastI-factor leader sequence at the 5′ end of the Hu-Asp-encoding nucleotidesequence.

Insect host cell culture systems may also be used for the expression ofHu-Asp polypeptides. In a preferred embodiment, the Hu-Asp polypeptidesof the invention are expressed using an insect cell expression system(see Example 10). Additionally, a baculovirus expression system can beused for expression in insect cells as reviewed by Luckow and Summers,Bio/Technology 6:47 (1988).

In another preferred embodiment, the Hu-Asp polypeptide is expressed inmammalian host cells. Nonlimiting examples of suitable mammalian celllines include the COS7 line of monkey kidney cells (Gluzman et al., Cell23:175 (1981)), human embyonic kidney cell line 293, and Chinese hamsterovary (CHO) cells. Preferably, Chinese hamster ovary (CHO) cells areused for expression of Hu-Asp proteins (Example 11).

The choice of a suitable expression vector for expression of the Hu-Asppolypeptides of the invention will of course depend upon the specificmammalian host cell to be used, and is within the skill of the ordinaryartisan. Examples of suitable expression vectors include pcDNA3(Invitrogen) and pSVL (Pharmacia Biotech). A preferred vector forexpression of Hu-Asp polypeptides is pcDNA3.1-Hygro (Invitrogen).Expression vectors for use in mammalian host cells may includetranscriptional and translational control sequences derived from viralgenomes. Commonly used promoter sequences and enhancer sequences whichmay be used in the present invention include, but are not limited to,those derived from human cytomegalovirus (CMV), Adenovirus 2, Polyomavirus, and Simian virus 40 (SV40). Methods for the construction ofmammalian expression vectors are disclosed, for example, in Okayama andBerg (Mol. Cell. Biol. 3:280 (1983)); Cosman et al. (Mol. Immunol.23:935 (1986)); Cosman et al. (Nature 312:768 (1984)); EP-A-0367566; andWO 91/18982.

The polypeptides of the present invention may also be used to raisepolyclonal and monoclonal antibodies, which are useful in diagnosticassays for detecting Hu-Asp polypeptide expression. Such antibodies maybe prepared by conventional techniques. See, for example, Antibodies: ALaboratory Manual, Harlow and Land (eds.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (1988); Monoclonal Antibodies,Hybridomas: A New Dimension in Biological Analyses, Kennet et al.(eds.), Plenum Press, New York (1980). Synthetic peptides comprisingportions of Hu-Asp containing 5 to 20 amino acids may also be used forthe production of polyclonal or monoclonal antibodies after linkage to asuitable carrier protein including but not limited to keyhole limpethemacyanin (KLH), chicken ovalbumin, or bovine serum albumin usingvarious cross-lining reagents including carbodimides, glutaraldehyde, orif the peptide contains a cysteine, N-methylmaleimide. A preferredpeptide for immunization when conjugated to KLH contains the C-terminusof Hu-Asp1 or Hu-Asp2 comprising QRRPRDPEVVNDESSLVRHRWK (SEQ ID NO: 2,residues 497-518) or LRQQHDDFADDISLLK (SEQ ID NO:4, residues 486-501),respectively. See SEQ ID Nos. 33-34.

The Hu-Asp nucleic acid molecules of the present invention are alsovaluable for chromosome identification, as they can hybridize with aspecific location on a human chromosome. Hu-Asp1 has been localized tochromosome 21, while Hu-Asp2 has been localized to chromosome11q23.3-24.1. There is a current need for identifying particular siteson the chromosome, as few chromosome marking reagents based on actualsequence data (repeat polymorphisms) are presently available for markingchromosomal location. Once a sequence has been mapped to a precisechromosomal location, the physical position of the sequence on thechromosome can be correlated with genetic map data. The relationshipbetween genes and diseases that have been mapped to the same chromosomalregion can then be identified through linkage analysis, wherein thecoinheritance of physically adjacent genes is determined. Whether a geneappearing to be related to a particular disease is in fact the cause ofthe disease can then be determined by comparing the nucleic acidsequence between affected and unaffected individuals.

In another embodiment, the invention relates to a method of assayingHu-Asp function, specifically Hu-Asp2 function which involves incubatingin solution the Hu-Asp polypeptide with a suitable substrate includingbut not limited to a synthetic peptide containing the β-secretasecleavage site of APP, preferably one containing the mutation found in aSwedish kindred with inherited AD in which KM is changed to NL, suchpeptide comprising the sequence SEVNLDAEFR (SEQ ID NO:63) in an acidicbuffering solution, preferably an acidic buffering solution of pH5.5(see Example 12) using cleavage of the peptide monitored by highperformance liquid chromatography as a measure of Hu-Asp proteolyticactivity. Preferred assays for proteolytic activity utilize internallyquenched peptide assay substrates. Such suitable substrates includepeptides which have attached a paired flurophore and quencher includingbut not limited to 7-amino4-methyl coumarin and dinitrophenol,respectively, such that cleavage of the peptide by the Hu-Asp results inincreased fluorescence due to physical separation of the flurophore andquencher. Other paired flurophores and quenchers includebodipy-tetramethylrhodamine and QSY-5 (Molecular Probes, Inc.). In avariant of this assay, biotin or another suitable tag may be placed onone end of the peptide to anchor the peptide to a substrate assay plateand a flurophore may be placed at the other end of the peptide. Usefulflurophores include those listed above as well as Europium labels suchas W8044 (EG&g Wallac, Inc.). Cleavage of the peptide by Asp2 willrelease the flurophore or other tag from the plate, allowing compoundsto be assayed for inhibition of Asp2 proteolytic cleavage as shown by anincrease in retained fluorescence. Preferred colorimetric assays ofHu-Asp proteolytic activity utilize other suitable substrates thatinclude the P2 and P1 amino acids comprising the recognition site forcleavage linked to o-nitrophenol through an amide linkage, such thatcleavage by the Hu-Asp results in an increase in optical density afteraltering the assay buffer to alkaline pH.

In another embodiment, the invention relates to a method for theidentification of an agent that increases the activity of a Hu-Asppolypeptide selected from the group consisting of Hu-Asp1, Hu-Asp2(a),and Hu-Asp2(b), the method comprising

(a) determining the activity of said Hu-Asp polypeptide in the presenceof a test agent and in the absence of a test agent; and

(b) comparing the activity of said Hu-Asp polypeptide determined in thepresence of said test agent to the activity of said Hu-Asp polypeptidedetermined in the absence of said test agent;

whereby a higher level of activity in the presence of said test agentthan in the absence of said test agent indicates that said test agenthas increased the activity of said Hu-Asp polypeptide. Such tests can beperformed with Hu-Asp polypeptide in a cell free system and withcultured cells that express Hu-Asp as well as variants or isoformsthereof.

In another embodiment, the invention relates to a method for theidentification of an agent that decreases the activity of a Hu-Asppolypeptide selected from the group consisting of Hu-Asp1, Hu-Asp2(a),and Hu-Asp2(b), the method comprising

(a) determining the activity of said Hu-Asp polypeptide in the presenceof a test agent and in the absence of a test agent; and

(b) comparing the activity of said Hu-Asp polypeptide determined in thepresence of said test agent to the activity of said Hu-Asp polypeptidedetermined in the absence of said test agent;

whereby a lower level of activity in the presence of said test agentthan in the absence of said test agent indicates that said test agenthas decreased the activity of said Hu-Asp polypeptide. Such tests can beperformed with Hu-Asp polypeptide in a cell free system and withcultured cells that express Hu-Asp as well as variants or isoformsthereof.

In another embodiment, the invention relates to a novel cell line(HEK125.3 cells) for measuring processing of amyloid β peptide (Aβ) fromthe amyloid protein precursor (APP). The cells are stable transformantsof human embryonic kidney 293 cells (HEK293) with a bicistronic vectorderived from pIRES-EGFP (Clontech) containing a modified human APP cDNA,an internal ribosome entry site and an enhanced green fluorescentprotein (EGFP) cDNA in the second cistron. The APP cDNA was modified byadding two lysine codons to the carboxyl terminus of the APP codingsequence. This increases processing of Aβ peptide from human APP by 2-4fold. This level of Aβ peptide processing is 60 fold higher than is seenin nontransformed HEK293 cells. HEK125.3 cells will be useful for assaysof compounds that inhibit Aβ peptide processing. This invention alsoincludes addition of two lysine residues to the C-terminus of other APPisoforms including the 751 and 770 amino acid isoforms, to isoforms ofAPP having mutations found in human AD including the Swedish KM→NL andV717-F mutations, to C-terminal fragments of APP, such as thosebeginning with the β-secretase cleavage site, to C-terminal fragments ofAPP containing the β-secretase cleavage site which have been operablylinked to an N-terminal signal peptide for membrane insertion andsecretion, and to C-terminal fragments of APP which have been operablylinked to an N-terminal signal peptide for membrane insertion andsecretion and a reporter sequence including but not limited to greenfluorescent protein or alkaline phosphatase, such that β-secretasecleavage releases the reporter protein from the surface of cellsexpressing the polypeptide.

Having generally described the invention, the same win be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLE 1 Development of a Search Algorithm Useful for theIdentification of Aspartyl Proteases, and Identification of C. elegansAspartyl Protease Genes in Wormpep 12

Materials and Methods

Classical aspartyl proteases such as pepsin and renin possess atwo-domain structure which folds to bring two aspartyl residues intoproximity within the active site. These are embedded in the shorttripeptide motif DTG, or more rarely, DSG. The DTG or DSG active sitemotif appears at about residue 25-30 in the enzyme, but at about 65-70in the proenzyme (prorenin, pepsinogen). This motif appears again about150-200 residues downstream. The proenzyme is activated by cleavage ofthe N-terminal prodomain. This pattern exemplifies the double domainstructure of the modern day aspartyl enzymes which apparently arose bygene duplication and divergence. Thus;

NH₂------------X------D²⁵TG---------------Y-------D^(Y+25)TG----------------C

where X denotes the beginning of the enzyme, following the N-terminalprodomain, and Y denotes the center of the molecule where the generepeat begins again.

In the case of the retroviral enzymes such as the HIV protease, theyrepresent only a half of the two-domain structures of well-known enzymeslike pepsin, cathepsin D, renin, etc. They have no prosegment, but arecarved out of a polyprotein precursor containing the gag and polproteins of the virus. They can be represented by:

NH₂---------D²⁵TG---------------------C100

This “monomer” only has about 100 aa, so is extremely parsimonious ascompared to the other aspartyl protease “dimers” which have of the orderof 330 or so aa, not counting the N-terminal prodomain.

The limited length of the eukaryotic aspartyl protease active site motifmakes it difficult to search EST collections for novel sequences. ESTsequences typically average 250 nucleotides, and so in this case wouldbe unlikely to span both aspartyl protease active site motifs. Instead,we turned to the C. elegans genome. The C. elegans genome is estimatedto contain around 13,000 genes. Of these, roughly 12,000 have beensequenced and the corresponding hypothetical open reading frame (ORF)has been placed in the database Wormpep12. We used this database as thebasis for a whole genome scan of a higher eukaryote for novel aspartylproteases, using an algorithm that we developed specifically for thispurpose. The following AWK script for locating proteins containing twoDTG or DSG motifs was used for the search, which was repeated four timesto recover all pairwise combinations of the aspartyl motif.

BEGIN{RS=“>”} /* defines “>” as record separator for FASTA format */ {pos = index($0,“DTG”) /*finds “DTG” in record*/ if (pos>0) { rest =substr($0,pos+3) /*get rest of record after first DTG*/ pos2 =index(rest,“DTG”) /*find second DTG*/ if (pos2>0) printf (“%s%s\n”, “>”,$0) } /*report hits*/ } }

The AWK script shown above was used to search Wormpep12, which wasdownloaded from ftp.sanger.ac.uk/pub/databases/wormpep, for sequenceentries containing at least two DTG or DSG motifs. Using AWK limitedeach record to 3000 characters or less. Thus, 35 or so larger recordswere eliminated manually from Wormpep12 as in any case these wereunlikely to encode aspartyl proteases.

Results and Discussion

The Wormpep 12 database contains 12,178 entries, although some of these(<10%) represent alternatively spliced transcripts from the same gene.Estimates of the number of genes encoded in the C. elegans genome is onthe order of 13,000 genes, so Wormpep12 may be estimated to covergreater than 90% of the C. elegans genome.

Eukaryotic aspartyl proteases contain a two-domain structure, probablyarising from ancestral gene duplication. Each domain contains the activesite motif D(S/T)G located from 20-25 amino acid residues into eachdomain. The retroviral (e.g., HIV protease) or retrotransposon proteasesare homodimers of subunits which are homologous to a single eukaryoticaspartyl protease domain. An AWK script was used to search the Wormpep12database for proteins in which the D(S/T)G motif occurred at leasttwice. This identified >60 proteins with two DTG or DSG motifs. Visualinspection was used to select proteins in which the position of theaspartyl domains was suggestive of a two-domain structure meeting thecriteria described above.

In addition, the PROSITE eukaryotic and viral aspartyl protease activesite pattern PS00141 was used to search Wormpep12 for candidate aspartylproteases. (Bairoch A., Bucher P., Hofmann K., The PROSITE database: itsstatus in 1997, Nucleic Acids Res. 24:217-221(1997)). This generated anoverlapping set of Wormpep12 sequences. Of these, seven sequencescontained two DTG or DSG motifs and the PROSITE aspartyl protease activesite pattern. Of these seven, three were found in the same cosmid clone(F21F8.3, F21F8.4, and F21F8.7) suggesting that they represent a familyof proteins that arose by ancestral gene duplication. Two other ORFswith extensive homology to F21F8.3, F21F8.4 and F21F8.7 are present inthe same gene cluster (F21F8.2 and F21F8.6), however, these contain onlya single DTG motif. Exhaustive BLAST searches with these seven sequencesagainst Wormpep12 failed to reveal additional candidate aspartylproteases in the C. elegans genome containing two repeats of the DTG orDSG motif.

BLASTX search with each C. elegans sequence against SWISS-PROT, GenPepand TREMBL revealed that R12H7.2 was the closest worm homologue to theknown mammalian aspartyl proteases, and that T18H9.2 was somewhat moredistantly related, while CEASP1, F21F8.3, F21F8.4, and F21F8.7 formed asubcluster which had the least sequence homology to the mammaliansequences.

Discussion

APP, the presenilins, and p35, the activator of cdk5, all undergointracellular proteolytic processing at sites which conform to thesubstrate specificity of the HIV protease. Dysregulation of a cellularaspartyl protease with the same substrate specificity, might thereforeprovide a unifying mechanism for causation of the plaque and tanglepathologies in AD. Therefore, we sought to identify novel human aspartylproteases. A whole genome scan in C. elegans identified seven openreading frames that adhere to the aspartyl protease profile that we hadidentified. These seven aspartyl proteases probably comprise thecomplete complement of such proteases in a simple, multicellulareukaryote. These include four closely related aspartyl proteases uniqueto C. elegans which probably arose by duplication of an ancestral gene.The other three candidate aspartyl proteases (T18H9.2, R12H7.2 andC11D2.2) were found to have homology to mammalian gene sequences.

EXAMPLE 2 Identification of Novel Human Aspartyl Proteases UsingDatabase Mining by Genome Bridging

Materials and Methods

Computer-assisted Analysis of EST Databases, cDNA, and PredictedPolypeptide Sequences

Exhaustive homology searches of EST databases with the CEASP1, F21F8.3,F21F8.4, and F21F8.7 sequences failed to reveal any novel mammalianhomologues. TBLASTN searches with R12H7.2 showed homology to cathepsinD, cathepsin E, pepsinogen A, pepsinogen C and renin, particularlyaround the DTG motif within the active site, but also failed to identifyany additional novel mammalian aspartyl proteases. This indicates thatthe C. elegans genome probably contains only a single lysosomal aspartylprotease which in mammals is represented by a gene family that arosethrough duplication and consequent modification of an ancestral gene.

TBLASTN searches with T18H9.2, the remaining C. elegans sequence,identified several ESTs which assembled into a contig encoding a novelhuman aspartyl protease (Hu-ASP1). As is described above in Example 1,BLASTX search with the Hu-ASP1 contig against SWISS-PROT revealed thatthe active site motifs in the sequence aligned with the active sites ofother aspartyl proteases. Exhaustive, repetitive rounds of BLASTNsearches against LifeSeq, LifeSeqFL, and the public EST collectionsidentified 102 EST from multiple cDNA libraries that assembled into asingle contig. The 51 sequences in this contig found in public ESTcollections also have been assembled into a single contig (THC213329) byThe Institute for Genome Research (TIGR). The TIGR annotation indicatesthat they failed to find any hits in the database for the contig. Notethat the TIGR contig is the reverse complement of the LifeSeq contigthat we assembled. BLASTN search of Hu-ASP1 against the rat and mouseEST sequences in ZooSeq revealed one homologous EST in each database(Incyte clone 700311523 and IMAGE clone 313341, GenBank accession numberW10530, respectively).

TBLASTN searches with the assembled DNA sequence for Hu-ASP1 againstboth LifeSeqFL and the public EST databases identified a second, relatedhuman sequence-(Hu-Asp2) represented by a single EST (2696295).Translation of this partial cDNA sequence reveals a single DTG motifwhich has homology to the active site motif of a bovine aspartylprotease, NM1.

BLAST searches, contig assemblies and multiple sequence alignments wereperformed using the bioinformatics tools provided with the LifeSeq,LifeSeqFL and LifeSeq Assembled databases from Incyte. Predicted proteinmotifs were identified using either the ProSite dictionary (Motifs inGCG 9) or the Pfam database.

Full-length cDNA Cloning of Hu-Asp1

The open reading frame of C. elegans gene T18H9.2CE was used to queryIncyte LifeSeq and LifeSeq-FL databases and a single electronic assemblyreferred to as 1863920CE1 was detected. The 5′ most cDNA clone in thiscontig, 1863920, was obtained from Incyte and completely sequenced onboth strands. Translation of the open reading frame contained withinclone 1863920 revealed the presence of the duplicated aspartyl proteaseactive site motif (DTG/DSG) but the 5′ end was incomplete. The remainderof the Hu-Asp1 coding sequence was determined by 5′ Marathon RACEanalysis using a human placenta Marathon ready cDNA template (Clontech).A 3′-antisense oligonucleotide primer specific for the 5′ end of clone1863920 was paired with the 5′-sense primer specific for the Marathonready cDNA synthetic adaptor in the PCR. Specific PCR products weredirectly sequenced by cycle sequencing and the resulting sequenceassembled with the sequence of clone 1863920 to yield the completecoding sequence of Hu-Asp-1 (SEQ ID No. 1).

Several interesting features are present in the primary amino acidsequence of Hu-Asp1 (FIG. 1, SEQ ID No. 2). The sequence contains asignal peptide (residues 1-20 in SEQ ID No. 2), a pro-segment, and acatalytic domain containing two copies of the aspartyl protease activesite motif (DTG/DSG). The spacing between the first and second activesite motifs is about 200 residues which should correspond to theexpected size of a single, eukaryotic aspartyl protease domain. Moreinterestingly, the sequence contains a predicted transmembrane domain(residues 469-492 in SEQ ID No.2) near its C-terminus which suggeststhat the protease is anchored in the membrane. This feature is not foundin any other aspartyl protease.

Cloning of a Full-length Hu-Asp-2 cDNAs

As is described above in Example 1, genome wide scan of theCaenorhabditis elegans database Wormpep12 for putative aspartylproteases and subsequent mining of human EST databases revealed a humanortholog to the C. elegans gene T18H9.2 referred to as Hu-Asp1. Theassembled contig for Hu-Asp1 was used to query for human paralogs usingthe BLAST search tool in human EST databases and a single significantmatch (2696295CE1) with approximately 60% shared identity was found inthe LifeSeq FL database. Similar queries of either gb105PubEST or thefamily of human databases available from TIGR did not identify similarEST clones. cDNA clone 2696295, identified by single pass sequenceanalysis from a human uterus cDNA library, was obtained from Incyte andcompletely sequence on both strands. This clone contained an incomplete1266 bp open-reading frame that encoded a 422 amino acid polypeptide butlacked an initiator ATG on the 5′ end. Inspection of the predictedsequence revealed the presence of the duplicated aspartyl proteaseactive site motif DTG/DSG, separated by 194 amino acid residues.Subsequent queries of later releases of the LifeSeq EST databaseidentified an additional ESTs, sequenced from a human astrocyte cDNAlibrary (4386993), that appeared to contain additional 5′ sequencerelative to clone 2696295. Clone 4386993 was obtained from Incyte andcompletely sequenced on both strands. Comparative analysis of clone4386993 and clone 2696295 confirmed that clone 4386993 extended theopen-reading frame by 31 amino acid residues including two in-frametranslation initiation codons. Despite the presence of the two in-frameATGs, no in-frame stop codon was observed upstream of the ATG indicatingthat the 4386993 may not be full-length. Furthermore, alignment of thesequences of clones 2696295 and 4386993 revealed a 75 base pairinsertion in clone 2696295 relative to clone 4386993 that results in theinsertion of 25 additional amino acid residues in 2696295. The remainderof the Hu-Asp2 coding sequence was determined by 5′ Marathon RACEanalysis using a human hippocampus Marathon ready cDNA template(Clontech). A 3′-antisense oligonucleotide primer specific for theshared 5′-region of clones 2696295 and 4386993 was paired with the5′-sense primer specific for the Marathon ready cDNA synthetic adaptorin the PCR. Specific PCR products were directly sequenced by cyclesequencing and the resulting sequence assembled with the sequence ofclones 2696295 and 4386993 to yield the complete coding sequence ofHu-Asp2(a) (SEQ ID No. 3) and Hu-Asp2(b) (SEQ ID No. 5), respectively.

Several interesting features are present in the primary amino acidsequence of Hu-Asp2(a) (FIG. 3 and SEQ ID No. 4) and Hu-Asp-2(b) (FIG.2, SEQ ID No. 6). Both sequences contain a signal peptide (residues 1-21in SEQ ID No. 4 and SEQ ID No. 6), a pro-segment, and a catalytic domaincontaining two copies of the aspartyl protease active site motif(DTG/DSG). The spacing between the first and second active site motifsis variable due to the 25 amino acid residue deletion in Hu-Asp-2(b) andconsists of 168-versus-194 amino acid residues, for Hu-Asp2(b) andHu-Asp-2(a), respectively. More interestingly, both sequences contains apredicted transmembrane domain (residues 455-477 in SEQ ID No.4 and430-452 in SEQ ID No. 6) near their C-termini which indicates that theprotease is anchored in the membrane. This feature is not found in anyother aspartyl protease except Hu-Asp1.

EXAMPLE 3 Molecular Cloning of Mouse Asp2 cDNA and Genomic DNA

Cloning and Characterization of Murine Asp2 cDNA

The murine ortholog of Hu-Asp2 was cloned using a combination of cDNAlibrary screening, PCR, and genomic cloning. Approximately 500,000independent clones from a mouse brain cDNA library were screened using a³²P-labeled coding sequence probe prepared from Hu-Asp2. Replicatepositives were subjected to DNA sequence analysis and the longest cDNAcontained the entire 3′ untranslated region and 47 amino acids in thecoding region. PCR amplification of the same mouse brain cDNA librarywith an antisense oligonucleotide primer specific for the 5′-most cDNAsequence determined above and a sense primer specific for the 5′ regionof human Asp2 sequence followed by DNA sequence analysis gave anadditional 980 bp of the coding sequence. The remainder of the 5′sequence of murine Asp-2 was derived from genomic sequence (see below).

Isolation and Sequence Analysis of the Murine Asp-2 Gene

A murine EST sequence encoding a portion of the murine Asp2 cDNA wasidentified in the GenBank EST database using the BLAST search tool andthe Hu-Asp2 coding sequence as the query. Clone g3160898 displayed 88%shared identity to the human sequence over 352 bp. Oligonucleotideprimer pairs specific for this region of murine Asp2 were thensynthesized and used to amplify regions of the murine gene. Murinegenomic DNA, derived from strain 129/SvJ, was amplified in the PCR (25cycles) using various primer sets specific for murine Asp2 and theproducts analyzed by agarose gel electrophoresis. The primer set Zoo-1and Zoo-4 amplified a 750 bp fragment that contained approximately 600bp of intron sequence based on comparison to the known cDNA sequence.This primer set was then used to screen a murine BAC library by PCR, asingle genomic clone was isolated and this cloned was confirmed containthe murine Asp2 gene by DNA sequence analysis. Shotgun DNA sequencing ofthis Asp2 genomic clone and comparison to the cDNA sequences of bothHu-Asp2 and the partial murine cDNA sequences defined the full-lengthsequence of murine Asp2 (SEQ ID No. 7). The predicted amino acidsequence of murine Asp2 (SEQ ID No. 8) showed 96.4% shared identity (GCGBestFit algorithm) with 18/501 amino acid residue substitutions comparedto the human sequence (FIG. 4). The proteolytic processing of murineAsp2(a) is believed to be analogous to the processing described abovefor human Asp2(a). In addition, a variant lacking amino acid residues190-214 of SEQ ID NO: 8 is specifically contemplated as a murine Asp2(b)polypeptide and is set out as SEQ ID NO: 73. All forms of murine Asp2(b)gene and protein are intended as aspects of the invention.

EXAMPLE 4 Tissue Distribution of Expression of Hu-Asp2 Transcripts

Materials and Methods

The tissue distribution of expression of Hu-Asp-2 was determined usingmultiple tissue Northern blots obtained from Clontech (Palo Alto,Calif.). Incyte clone 2696295 in the vector pINCY was digested tocompletion with EcoRI/NotI and the 1.8 kb cDNA insert purified bypreparative agarose gel electrophoresis. This fragment was radiolabeledto a specific activity >1×10⁹ dpm/μg by random priming in the presenceof [(α-³²P-dATP] (>3000 Ci/mmol, Amersham, Arlington Heights, Ill.) andKlenow fragment of DNA polymerase I. Nylon filters containing denatured,size fractionated poly A⁺ RNAs isolated from different human tissueswere hybridized with 2×10⁶ dpm/ml probe in ExpressHyb buffer (Clontech,Palo Alto, Calif.) for 1 hour at 68° C. and washed as recommended by themanufacture. Hybridization signals were visualized by autoradiographyusing BioMax XR film (Kodak, Rochester, N.Y.) with intensifying screensat −80° C.

Results and Discussion

Limited information on the tissue distribution of expression of Hu-Asp-2transcripts was obtained from database analysis due to the relativelysmall number of ESTs detected using the methods described above (<5). Inan effort to gain further information on the expression of the Hu-Asp2gene, Northern analysis was employed to determine both the size(s) andabundance of Hu-Asp2 transcripts. PolyA⁺ RNAs isolated from a series ofperipheral tissues and brain regions were displayed on a solid supportfollowing separation under denaturing conditions and Hu-Asp2 transcriptswere visualized by high stringency hybridization to radiolabeled insertfrom clone 2696295. The 2696295 cDNA probe visualized a constellation oftranscripts that migrated with apparent sizes of 3.0 kb, 4.4 kb and 8.0kb with the latter two transcript being the most abundant.

Across the tissues surveyed, Hu-Asp2 transcripts were most abundant inpancreas and brain with lower but detectable levels observed in allother tissues examined except thymus and PBLs. Given the relativeabundance of Hu-Asp2 transcripts in brain, the regional expression inbrain regions was also established. A similar constellation oftranscript sizes were detected in all brain regions examined[cerebellum, cerebral cortex, occipital pole, frontal lobe, temporallobe and putamen] with the highest abundance in the medulla and spinalcord.

EXAMPLE 5 Northern Blot Detection of HuAsp-1 and HuAsp-2 Transcripts inHuman Cell Lines

A variety of human cell lines were tested for their ability to produceHu-Asp1 and Asp2 mRNA. Human embryonic kidney (HEK-293) cells, Africangreen monkey (Cos-7) cells, Chinese hamster ovary (CHO) cells, HELAcells, and the neuroblastoma cell line IMR-32 were all obtained from theATCC. Cells were cultured in DME containing 10% FCS except CHO cellswhich were maintained in α-MEM/10% FCS at 37° C. in 5% CO₂ until theywere near confluence. Washed monolayers of cells (3×10⁷) were lysed onthe dishes and poly A⁺ RNA extracted using the Qiagen Oligotex DirectmRNA kit. Samples containing 2 μg of poly A⁺ RNA from each cell linewere fractionated under denaturing conditions (glyoxal-treated),transferred to a solid nylon membrane support by capillary action, andtranscripts visualized by hybridization with random-primed labeled (³²p)coding sequence probes derived from either Hu-Asp1 or Hu-Asp2.Radioactive signals were detected by exposure to X-ray film and by imageanalysis with a PhosphorImager.

The Hu-Asp1 cDNA probe visualized a similar constellation of transcripts(2.6 kb and 3.5 kb) that were previously detected is human tissues. Therelative abundance determined by quantification of the radioactivesignal was Cos-7>HEK 292=HELA>IMR32.

The Hu-Asp2 cDNA probe also visualized a similar constellation oftranscripts compared to tissue (3.0 kb, 4.4 kb, and 8.0 kb) with thefollowing relative abundance; HEK 293>Cos 7>MR32>HELA.

EXAMPLE 6 Modification of APP to Increase Aβ Processing for in vitroScreening

Human cell lines that process Aβ peptide from APP provide a means toscreen in cellular assays for inhibitors of β- and γ-secretase.Production and release of Aβ peptide into the culture supernatant ismonitored by an enzyme-linked immunosorbent assay (EIA). Althoughexpression of APP is widespread and both neural and non-neuronal celllines process and release Aβ peptide, levels of endogenous APPprocessing are low and difficult to detect by EIA. Aβ processing can beincreased by expressing in transformed cell lines mutations of APP thatenhance Aβ processing. We made the serendipitous observation thataddition of two lysine residues to the carboxyl terminus of APP695increases Aβ processing still further. This allowed us to create atransformed cell line that releases Aβ peptide into the culture mediumat the remarkable level of 20,000 pg/ml.

Materials and Methods

Materials

Human embryonic kidney cell line 293 (HEK293 cells) were obtainedinternally. The vector pIRES-EGFP was purchased from Clontech.Oligonucleotides for mutation using the polymerase chain reaction (PCR)were purchased from Genosys. A plasmid containing human APP695 (SEQ IDNo. 9 [nucleotide] and SEQ ID No. 10 [amino acid]) was obtained fromNorthwestern University Medical School. This was subcloned into pSK(Stratagene) at the Not1 site creating the plasmid pAPP695.

Mutagenesis Protocol

The Swedish mutation (K670N, M671L) was introduced into pAPP695 usingthe Stratagene Quick Change Mutagenesis Kit to create the plasmidpAPP695NL (SEQ ID No. 11 [nucleotide] and SEQ ID No. 12 [amino acid]).To introduce a di-lysine motif at the C-terminus of APP695, the forwardprimer #276 5′ GACTGACCACTCGACCAGGTTC (SEQ ID No. 47) was used with the“patch” primer #274 5′CGAATTAAATTCCAGCACACTGGCTACTTCTTGTTCTGCATCTCAAAGAAC (SEQ ID No. 48) andthe flanking primer #275 CGAATTAAATTCCAGCACACTGGCTA (SEQ ID No. 49) tomodify the 3′ end of the APP695 cDNA (SEQ ID No. 15 [nucleotide] and SEQID No. 16 [amino acid]). This also added a BstX1 restriction site thatwill be compatible with the BstX1 site in the multiple cloning site ofpIRES-EGFP. PCR amplification was performed with a Clontech HF AdvantagecDNA PCR kit using the polymerase mix and buffers supplied by themanufacturer. For “patch” PCR, the patch primer was used at 1/20th themolar concentration of the flanking primers. PCR amplification productswere purified using a QIAquick PCR purification kit (Qiagen). Afterdigestion with restriction enzymes, products were separated on 0.8%agarose gels and then excised DNA fragments were purified using aQIAquick gel extraction kit (Qiagen).

To reassemble a modified APP695-Sw cDNA, the 5′ Not1-Bgl2 fragment ofthe APP695-Sw cDNA and the 3′ Bgl2-BstX1 APP695 cDNA fragment obtainedby PCR were ligated into pIRES-EGFP plasmid DNA opened at the Not1 andBstX1 sites. Ligations were performed for 5 minutes at room temperatureusing a Rapid DNA Ligation kit (Boehringer Mannheim) and transformedinto Library Efficiency DH5a Competent Cells (GibcoBRL LifeTechnologies). Bacterial colonies were screened for inserts by PCRamplification using primers #276 and #275. Plasmid DNA was purified formammalian cell transfection using a QIAprep Spin Miniprep kit (Qiagen).The construct obtained was designated pMG125.3 (APPSW-KK, SEQ ID No. 17[nucleotide] and SEQ ID No. 18 [amino acid]).

Mammalian Cell Transfection

HEK293 cells for transfection were grown to 80% confluence in Dulbecco'smodified Eagle's medium (DMEM) with 10% fetal bovine serum.Cotransfections were performed using LipofectAmine (Gibco-BRL) with 3 μgpMG125.3 DNA and 9 μg pcDNA3.1 DNA per 10×10⁶ cells. Three daysposttransfection, cells were passaged into medium containing G418 at aconcentration of 400 μg/ml. After three days growth in selective medium,cells were sorted by their fluorescence.

Clonal Selection of 125.3 Cells by FACS

Cell samples were analyzed on an EPICS Elite ESP flow cytometer(Coulter, Hialeah, Fla.) equipped with a 488 nm excitation line suppliedby an air-cooled argon laser. EGFP emission was measured through a 525nm band-pass filter and fluorescence intensity was displayed on a4-decade log scale after gating on viable cells as determined by forwardand right angle light scatter. Single green cells were separated intoeach well of one 96 well plate containing growth medium without G418.After a four day recovery period, G418 was added to the medium to afinal concentration of 400 μg/ml. After selection, 32% of the wellscontained expanding clones. Wells with clones were expanded from the 96well plate to a 24 well plate and then a 6 well plate with the fastestgrowing colonies chosen for expansion at each passage. The final cellline selected was the fastest growing of the final six passaged. Thisclone, designated 125.3, has been maintained in G418 at 400 ug/ml withpassage every four days into fresh medium. No loss of Aβ production ofEGFP fluorescence has been seen over 23 passages.

Aβ EIA Analysis (Double Antibody Sandwich ELISA for hAβ 1-40/42)

Cell culture supernatants harvested 48 hours after transfection wereanalyzed in a standard Aβ EIA as follows. Human Aβ 1-40 or 1-42 wasmeasured using monoclonal antibody (mAb) 6E10 (Senetek, St. Louis, Mo.)and biotinylated rabbit antiserum 162 or 164 (New York State Institutefor Basic Research, Staten Island, N.Y.) in a double antibody sandwichELISA. The capture antibody 6E10 is specific to an epitope present onthe N-terminal amino acid residues 1-16 of hAβ. The conjugated detectingantibodies 162 and 164 are specific for hAβ 1-40 and 1-42, respectively.Briefly, a Nunc Maxisorp 96 well immunoplate was coated with 100 μl/wellof mAb 6E10 (5 μg/ml) diluted in 0.1M carbonate-bicarbonate buffer, pH9.6 and incubated at 4° C. overnight. After washing the plate 3× with0.01M DPBS (Modified Dulbecco's Phosphate Buffered Saline (0.008M sodiumphosphate, 0.002M potassium phosphate, 0.14M sodium chloride, 0.01 Mpotassium chloride, pH 7.4) from Pierce, Rockford, Ill.) containing0.05% of Tween-20 (DPBST), the plate was blocked for 60 minutes with 200μl of 10% normal sheep serum (Sigma) in 0.01M DPBS to avoid non-specificbinding. Human Aβ 1-40 or 1-42 standards 100 μl/well (Bachem, Torrance,Calif.) diluted, from a 1 mg/ml stock solution in DMSO, in culturemedium was added after washing the plate, as well as 100 μl/well ofsample, e.g., conditioned medium of transfected cells.

The plate was incubated for 2 hours at room temperature and 4° C.overnight. The next day, after washing the plate, 100 μl/wellbiotinylated rabbit antiserum 162 1:400 or 164 1:50 diluted inDPBST+0.5% BSA was added and incubated at room temperature for 1 hour,15 minutes. Following washes, 100 μl/well neutravidin-horseradishperoxidase (Pierce, Rockford, Ill.) diluted 1:10,000 in DPBST wasapplied and incubated for 1 hour at room temperature. After the lastwashes 100 μl/well of o-phenylnediamine dihydrochloride (SigmaChemicals, St. Louis, Mo.) in 5.0 mM citric acid/100 mM sodium phosphatebuffer (Sigma Chemicals, St. Louis, Mo.), pH 5.0, was added as substrateand the color development was monitored at 450 nm in a kineticmicroplate reader for 20 minutes using Soft max Pro software. Allstandards and samples were run in triplicates. The samples withabsorbance values falling within the standard curve were extrapolatedfrom the standard curves using Soft max Pro software and expressed inpg/ml culture medium.

Results

Addition of two lysine residues to the carboxyl terminus of APP695greatly increases Aβ processing in HEK293 cells as shown by transientexpression (Table 1). Addition of the di-lysine motif to APP695increases Aβ processing to that seen with the APP695 containing theSwedish mutation. Combining the di-lysine motif with the Swedishmutation further increases processing by an additional 2.8 fold.

Cotransformation of HEK293 cells with pMG125.3 and pcDNA3.1 allowed dualselection of transformed cells for G418 resistance and high levelexpression of EGFP. After clonal selection by FACS, the cell lineobtained, produces a remarkable 20,000 pg Aβ peptide per ml of culturemedium after growth for 36 hours in 24 well plates. Production of Aβpeptide under various growth conditions is summarized in Table 2.

TABLE 1 Release of Aβ peptide into the culture medium 48 hours aftertransient transfection of HEK293 cells with the indicated vectorscontaining wildtype or modified APP. Values tabulated are mean ± SD andP-value for pairwise comparison using Student's t-test assuming unequalvariances. Aβ 1-40 peptide APP Construct (pg/ml) Fold Increase P-valuepIRES-EGFP vector 147 + 28 1.0 wt APP69S (142.3) 194 + 15 1.3 0.051 wtAPP695-KK (124.1) 424 + 34 2.8 3 × 10-5 APP695-Sw (143.3) 457 + 65 3.1 2× 10-3 APP695-SwKK (125.3) 1308 + 98  8.9 3 × 10-4

TABLE 2 Release of Aβ peptide from HEK125.3 cells under various growthconditions. Type of Culture Volume of Duration of Aβ 1-40 Aβ 1-42 PlateMedium Culture pg/ml) (pg/ml) 24 well plate 400 ul 36 hr 28,036 1,439

EXAMPLE 7 Antisense Oligomer Inhibition of Abeta Processing in HEK125.3Cells

The sequences of Hu-Asp1 and Hu-Asp2 were provided to Sequitur, Inc(Natick, Mass.) for selection of targeted sequences and design of 2ndgeneration chimeric antisense oligomers using prorietary technology(Sequitur Ver. D Pat pending #3002). Antisense oligomers Lot# S644,S645, S646 and S647 were targeted against Asp1. Antisense oligomers Lot#S648, S649, S650 and S651 were targeted against Asp2. Control antisenseoligomers Lot# S652, S653, S655, and S674 were targeted against anirrelevant gene and antisense oligomers Lot #S656, S657, S658, and S659were targeted against a second irrelevant gene.

For transfection with the antisense oligomers, HEK125.3 cells were grownto about 50% confluence in 6 well plates in Minimal Essential Medium(MEM) supplemented with 10% fetal calf serum. A stock solution ofoligofectin G (Sequitur Inc., Natick, Mass.) at 2 mg/ml was diluted to50 μg/ml in serum free MEM. Separately, the antisense oligomer stocksolution at 100 μM was diluted to 800 nM in Opti-MEM (GIBCO-BRL, GrandIsland, N.Y.). The diluted stocks of oligofectin G and antisenseoligomer were then mixed at a ratio of 1:1 and incubated at roomtemperature. After 15 minutes incubation, the reagent was diluted 10fold into MEM containing 10% fetal calf serum and 2 ml was added to eachwell of the 6 well plate after first removing the old medium. Aftertransfection, cells were grown in the continual presence of theoligofectin G/antisense oligomer. To monitor Aβ peptide release, 400 μlof conditioned medium was removed periodically from the culture well andreplaced with fresh medium beginning 24 hours after transfection. Aβpeptides in the conditioned medium were assayed via immunoprecipitationand Western blotting. Data reported are from culture supernatantsharvested 48 hours after transfection.

The 16 different antisense oligomers obtained from Sequitur Inc. weretransfected separately into HEK125.3 cells to determine their affect onAβ peptide processing. Only antisense oligomers targeted against Asp2significantly reduced Abeta processing by HEK125.3 cells. Both Aβ (1-40)and Aβ (1-42) were inhibited by the same degree. In Table 3, percentinhibition is calculated with respect to untransfected cells. Antisenseoligomer reagents giving greater than 50% inhibition are marked with anasterisk. For ASP2, 4 of 4 antisense oligomers gave greater than 50%inhibition with an average inhibition of 62% for Aβ 1-40 processing and60% for Aβ 1-42 processing.

TABLE 3 Inhibition of Aβ peptide release from HEK125.3 cells treatedwith antisense oligomers. Gene Targeted Antisense Oligomer Abeta (1-40)Abeta (1-42) Asp2-1 S648  71%*  67%* Asp2-2 S649  83%*  76%* Asp2-3 S650 46%*  50%* Asp2-4 S651  47%*  46%* Con1-1 S652 13% 18% Con1-2 S653 35%30% Con1-3 S655  9% 18% Con1-4 S674 29% 18% Con2-1 S656 12% 18% Con2-2S657 16% 19% Con2-3 S658  8% 35% Con2-4 S659  3% 18%

Since HEK293 cells derive from kidney, the experiment was extended tohuman IMR-32 neuroblastoma cells which express all three APP isoformsand which release Aβ peptides into conditioned medium at measurablelevels. [See Neill et al., J. NeuroSci. Res., (1994) 39: 482-93; andAsami-Odaka et al., Biochem., (1995) 34:10272-8.] Essentially identicalresults were obtained in the neuroblastoma cells as the HEK293 cells. Asshown in Table 3B, the pair of Asp2 antisense oligomers reduced Asp2mRNA by roughly one-half, while the pair of reverse control oligomerslacked this effect (Table 3B).

TABLE 3B Reduction of Aβ40 and Aβ42 in human neuroblastoma IMR-32 cellsand mouse neuroblastoma Neuro-2A cells treated with Asp2 antisense andcontrol oligomers as indicated. Oligomers were transfected inquadruplicate cultures. Values tabulated are normalized against culturestreated with oligofectin-G ™ only (mean + SD, ** p < 0.001 compared toreverse control oligomer). IMR-32 cells Neuro-2A cells Asp2 mRNA Aβ4Aβ42 Aβ40 Aβ42 Asp2-1A −75% −49 + 2%** −42 + −70 + −67 + 2%** 14%** 7%**Asp2-1R 0.16  −0 + 3%  21.26  −9 + 15%  1.05 Asp2-2A −39% −43 +0 3%**−44 + 18%** −61 + 12%** −61 + 12%** Asp2-2R 0.47  12.2  19.22  6.15 −8 + 10%

Together with the reduction in Asp2 mRNA there was a concomitantreduction in the release of Aβ40 and Aβ42 peptides into the conditionedmedium. Thus, Asp2 functions directly or indirectly in a human kidneyand a human neuroblastoma cell line to facilitate the processing of APPinto Aβ peptides. Molecular cloning of the mouse Asp2 cDNA revealed ahigh degree of homology to human (>96% amino acid identity, see Example3), and indeed, complete nucleotide identity at the sites targeted bythe Asp2-1A and Asp2-2A antisense oligomers. Similar results wereobtained in mouse Neuro-2a cells engineered to express APP-Sw-KK. TheAsp2 antisense oligomers reduced release of Aβ peptides into the mediumwhile the reverse control oligomers did not (Table 3B). Thus, the threeantisense experiments with HEK293, IMR-32 and Neuro-2a cells indicatethat Asp2 acts directly or indirectly to facilitate Aβ processing inboth somatic and neural cell lines.

EXAMPLE 8 Demonstration of Hu-Asp2 β-Secretase Activity in CulturedCells

Several mutations in APP associated with early onset Alzheimer's diseasehave been shown to alter Aβ peptide processing. These flank the—andC-terminal cleavage sites that release Aβ from APP. These cleavage sitesare referred to as the β-secretase and γ-secretase cleavage sites,respectively. Cleavage of APP at the β-secretase site creates aC-terminal fragment of APP containing 99 amino acids of 11,145 daltonsmolecular weight. The Swedish KM→NL mutation immediately upstream of theβ-secretase cleavage site causes a general increase in production ofboth the 1-40 and 1-42 amino acid forms of Aβ peptide. The London VFmutation (V717→F in the APP770 isoform) has little effect on total Aβpeptide production, but appears to preferentially increase thepercentage of the longer 1-42 amino acid form of Aβ peptide by affectingthe choice of β-secretase cleavage site used during APP processing.Thus, we sought to determine if these mutations altered the amount andtype of Aβ peptide produced by cultured cells cotransfected with aconstruct directing expression of Hu-Asp2.

Two experiments were performed which demonstrate Hu-Asp2 β-secretaseactivity in cultured cells. In the first experiment, treatment ofHEK125.3 cells with antisense oligomers directed against Hu-Asp2transcripts as described in Example 7 was found to decrease the amountof the C-terminal fragment of APP created by β-secretase cleavage(CTF99) (FIG. 9). This shows that Hu-Asp2 acts directly or indirectly tofacilitate β-secretase cleavage. In the second experiment, increasedexpression of Hu-Asp2 in transfected mouse Neuro2A cells is shown toincrease accumulation of the CTF99 β-secretase cleavage fragment (FIG.10). This increase is seen most easily when a mutant APP-KK clonecontaining a C-terminal di-lysine motif is used for transfection. Afurther increase is seen when Hu-Asp2 is cotransfected with APP-Sw-KKcontaining the Swedish mutation KM→NL. The Swedish mutation is known toincrease cleavage of APP by the β-secretase.

A second set of experiments demonstrate Hu-Asp2 facilitates γ-secretaseactivity in cotransfection experiments with human embryonic kidneyHEK293 cells. Cotransfection of Hu-Asp2 with an APP-KK clone greatlyincreases production and release of soluble Aβ1-40 and Aβ1-42 peptidesfrom HEK293 cells. There is a proportionately greater increase in therelease of Aβ1-42. A further increase in production of Aβ1-42 is seenwhen Hu-Asp2 is cotransfected with APP-VF (SEQ ID No. 13 [nucleotide]and SEQ ID No. 14 [amino acid]) or APP-VF-KK SEQ ID No. 19 [nucleotide]and SEQ ID No. 20 [amino acid]) clones containing the London mutationV717→F. The V717→F mutation is known to alter cleavage specificity ofthe APP γ-secretase such that the preference for cleavage at the Aβ42site is increased. Thus, Asp2 acts directly or indirectly to facilitateγ-secretase processing of APP at the β42 cleavage site.

Materials

Antibodies 6E10 and 4G8 were purchased from Senetek (St. Louis, Mo.).Antibody 369 was obtained from the laboratory of Paul Greengard at theRockefeller University. Antibody C8 was obtained from the laboratory ofDennis Selkoe at the Harvard Medical School and Brigham and Women'sHospital.

APP Constructs Used

The APP constructs used for transfection experiments comprised thefollowing

APP: wild-type APP695 (SEQ ID No. 9 and No. 10)

APP-Sw: APP695 containing the Swedish KM→NL mutation (SEQ ID No. 11 andNo. 12, wherein the lysine (K) at residue 595 of APP695 is changed toasparagine (N) and the methionine (M) at residue 596 of APP695 ischanged to leucine (L).),

APP-VF: APP695 containing the London V→F mutation (SEQ ID Nos. 13 & 14)(Affected residue 717 of the APP770 isoform corresponds with residue 642of the APP695 isoform. Thus, APP-VF as set in SEQ ID NO: 14 comprisesthe APP695 sequence, wherein the valine (V) at residue 642 is changed tophenylalanine (F).)

APP-KK: APP695 containing a C-terminal KK motif (SEQ ID Nos. 15 & 16),

APP-Sw-KK: APP695-Sw containing a C-terminal KK motif (SEQ ID No. 17 &18),

APP-VF-KK: APP695-VF containing a C-terminal KK motif (SEQ ID Nos. 19&20).

These were inserted into the vector pIRES-EGFP (Clontech, Palo AltoCalif.) between the Not1 and BstX1 sites using appropriate linkersequences introduced by PCR.

Transfection of Antisense Oligomers or Plasmid DNA Constructs in HEK293Cells, HEK125.3 cells and Neuro-2A Cells

Human embryonic kidney HEK293 cells and mouse Neuro-2a cells weretransfected with expression constructs using the Lipofectamine Plusreagent from Gibco/BRL. Cells were seeded in 24 well tissue cultureplates to a density of 70-80% confluence. Four wells per plate weretransfected with 2 μg DNA (3:1, APP:cotransfectant), 8 μl Plus reagent,and 4 μl Lipofectamine in OptiMEM. OptiMEM was added to a total volumeof 1 ml, distributed 200 μl per well and incubated 3 hours. Care wastaken to hold constant the ratios of the two plasmids used forcotransfection as well as the total amount of DNA used in thetransfection. The transfection media was replaced with DMEM, 10%FBS,NaPyruvate, with antibiotic/antimycotic and the cells were incubatedunder normal conditions (37° C., 5% CO₂) for 48 hours. The conditionedmedia were removed to polypropylene tubes and stored at −80° C. untilassayed for the content of Aβ1-40 and Aβ1-42 by EIA as described in thepreceding examples. Transfection of antisense oligomers into HEK125.3cells was as described in Example 7.

Preparation of Cell Extracts, Western Blot Protocol

Cells were harvested after being transfected with plasmid DNA for about60 hours. First, cells were transferred to 15-ml conical tube from theplate and centrifuged at 1,500 rpm for 5 minutes to remove the medium.The cell pellets were washed once with PBS. We then lysed the cells withlysis buffer (10 mM HEPES, pH 7.9, 150 mM NaCl, 10% glycerol, 1 mM EGTA,1 mM EDTA, 0.1 mM sodium vanadate and 1% NP-40). The lysed cell mixtureswere centrifuged at 5000 rpm and the supernatant was stored at −20° C.as the cell extracts. Equal amounts of extracts from HEK125.3 cellstransfected with the Asp2 antisense oligomers and controls wereprecipitated with antibody 369 that recognizes the C-terminus of APP andthen CTF99 was detected in the immunoprecipitate with antibody 6E10. Theexperiment was repeated using C8, a second precipitating antibody thatalso recognizes the C-terminus of APP. For Western blot of extracts frommouse Neuro-2a cells cotransfected with Hu-Asp2 and APP-KK, APP-Sw-KK,APP-VF-KK or APP-VF, equal amounts of cell extracts were electrophoresedthrough 4-10% or 10-20% Tricine gradient gels (NOVEX, San Diego,Calif.). Full length APP and the CTF99 β-secretase product were detectedwith antibody 6E10.

Results

Transfection of HEK125.3 cells with Asp2-1 or Asp2-2 antisense oligomersreduces production of the CTF β-secretase product in comparison to cellssimilarly transfected with control oligomers having the reverse sequence(Asp2-1 reverse & Asp2-2 reverse), see FIG. 9. Correspondingly,cotransfection of Hu-Asp2 into mouse Neuro-2a cells with the APP-KKconstruct increased the formation of CTF99. (See FIG. 10.) This wasfurther increased if Hu-Asp2 was coexpressed with APP-Sw-KK, a mutantform of APP containing the Swedish KM→NL mutation that increasesβ-secretase processing.

Effects of Asp2 on the production of Ab peptides from endogenouslyexpressed APP isoforms were assessed in HEK293 cells transfected with aconstruct expressing Asp2 or with the empty vector after selection oftransformants with the antibiotic G418. Aβ40 production was increased incells transformed with the Asp2 construct in comparison to thosetransformed with the empty vector DNA. Aβ40 levels in conditioned mediumcollected from the Asp2 transformed and control cultures was 424±45 pg/ml and 113±58 pg/ml, respectively (p<0.001). Aβ42 release was below thelimit of detection by the EIA, while the release of sAPPα wasunaffected, 112±8 ng/ml versus 111±40 ng/ml. This further indicates thatAsp2 acts directly or indirectly to facilitate the processing andrelease of Aβ from endogenously expressed APP.

Co-transfection of Hu-Asp2 with APP has little effect on Aβ40 productionbut increases Aβ42 production above background (Table 4). Addition ofthe di-lysine motif to the C-terminus of APP increases Aβ peptideprocessing about two fold, although Aβ40 and Aβ42 production remainquite low (352 pg/ml and 21 pg/ml, respectively). Cotransfection of Asp2with APP-KK further increases both Aβ40 and Aβ42 production.

The APP V717→F mutation has been shown to increase γ-secretaseprocessing at the Aβ42 cleavage site. Cotransfection of Hu-Asp2 with theAPP-VF or APP-VF-KK constructs increased Aβ42 production (a two foldincrease with APP-VF and a four-fold increase with APP-VF-KK, Table 4),but had mixed effects on Aβ40 production (a slight decrease with APP-VF,and a two fold increase with APP-VF-KK in comparison to the pcDNAcotransfection control. Thus, the effect of Asp2 on Aβ42 production wasproportionately greater leading to an increase in the ratio ofAβ42/total Ab. Indeed, the ratio of Aβ42/total Aβ reaches a very highvalue of 42% in HEK293 cells cotransfected with Hu-Asp2 and APP-VF-KK.

TABLE 4 Results of cotransfecting Hu-Asp2 or pcDNA plasmid DNA withvarious APP constructs containing the V717-F mutation that modifiesγ-secretase processing. Cotransfection with Asp2 consistently increasesthe ratio of Aβ42/total Aβ. Values tabulated are Aβ peptide pg/ml. pcDNAAsp2 Cotransfection Cotransfection Aβ40 Aβ42 Aβ42/Total Aβ40 Aβ42Aβ42/Total APP 192 ± 18 <4   <2% 188 ± 40  8 ± 10  3.9% APP-VF 118 ± 1515 ± 19 11.5% 85 ± 7  24 ± 12 22.4% APP-KK 352 ± 24 21 ± 6   5.5% 1062 ±101 226 ± 49 17.5% APP-VF-KK 230 ± 31 88 ± 24 27.7% 491 ± 35 355 ± 36  42%

EXAMPLE 9 Bacterial Expression of Human Asp2(a)

Expression of Recombinant Hu-Asp2(a) in E. coli

Hu-Asp2(a) can be expressed in E. coli after addition of N-terminalsequences such as a T7 tag (SEQ ID No. 21 and No. 22) or a T7 tagfollowed by a caspase 8 leader sequence (SEQ ID No. 23 and No. 24).Alternatively, reduction of the GC content of the 5′ sequence by sitedirected mutagenesis can be used to increase the yield of Hu-Asp2 (SEQID No. 25 and No. 26). In addition, Asp2(a) can be engineered with aproteolytic cleavage site (SEQ ID No. 27 and No. 28). To produce asoluble protein after expression and refolding, deletion of thetransmembrane domain and cytoplasmic tail, or deletion of the membraneproximal region, transmembrane domain, and cytoplasmic tail ispreferred. Any materials (vectors, host cells, etc.) and methodsdescribed herein to express Hu-Asp2(a) should in principle be equallyeffective for expression of Hu-Asp2(b).

Methods

PCR with primers containing appropriate linker sequences was used toassemble fusions of Asp2(a) coding sequence with N-terminal sequencemodifications including a T7 tag (SEQ ID Nos. 21 and 22) or a T7-caspase8 leader (SEQ ID Nos. 23 and 24). These constructs were cloned into theexpression vector pet23a(+) [Novagen] in which a T7 promoter directsexpression of a T7 tag preceding a sequence of multiple cloning sites.To clone Hu-Asp2 sequences behind the T7 leader of pet23a+, thefollowing oligonucleotides were used for amplification of the selectedHu-Asp2(a) sequence: #553=GTGGATCCACCCAGCACGGCATCCGGCTG (SEQ ID No. 35),#554=GAAAGCTTTCATGACTCATCTGTCTGTGGAATGTTG (SEQ ID No. 36) which placedBamHI and HindIII sites flanking the 5′ and 3′ ends of the insert,respectively. The Asp2(a) sequence was amplified from the full lengthAsp2(a) cDNA cloned into pcDNA3.1 using the Advantage-GC cDNA PCR[Clontech] following the manufacturer's supplied protocol usingannealing & extension at 68° C. in a two-step PCR cycle for 25 cycles.The insert and vector were cut with BamHI and HindIII, purified byelectrophoresis through an agarose gel, then ligated using the Rapid DNALigation kit [Boerhinger Mannheim]. The ligation reaction was used totransform the E. coli strain JM109 (Promega) and colonies were pickedfor the purification of plasmid (Qiagen,Qiaprep minispin) and DNAsequence analysis. For inducible expression using induction withisopropyl b-D-thiogalactopyranoside (IPTG), the expression vector wastransferred into E. coli strain BL21 (Statagene). Bacterial cultureswere grown in LB broth in the presence of ampicillin at 100 ug/ml, andinduced in log phase growth at an OD600 of 0.6-1.0 with 1 mM IPTG for 4hour at 37° C. The cell pellet was harvested by centrifugation.

To clone Hu-Asp2 sequences behind the T7 tag and caspase leader (SEQ IDNos. 23 and 24), the construct created above containing the T7-Hu-Asp2sequence (SEQ ID Nos. 21 and 22) was opened at the BamH1 site, and thenthe phosphorylated caspase 8 leader oligonucleotides#559=GATCGATGACTATCTCTGACTCTCCGCGTGAACAGGACG (SEQ ID No. 37),#560=GATCCGTCCTGTTCACGCGGAGAGTCAGAGATAGTCATC (SEQ ID No. 3 8) wereannealed and ligated to the vector DNA. The 5′ overhang for each set ofoligonucleotides was designed such that it allowed ligation into theBamHI site but not subsequent digestion with BamHI. The ligationreaction was transformed into JM109 as above for analysis of proteinexpression after transfer to E. coli strain BL21.

In order to reduce the GC content of the 5′ terminus of asp2(a), a pairof antiparallel oligos were designed to change degenerate codon bases in15 amino acid positions from G/C to A/T (SEQ ID Nos. 25 and 26). The newnucleotide sequence at the 5′ end of asp2 did not change the encodedamino acid and was chosen to optimize E. coli expression. The sequenceof the sense linker is 5′CGGCATCCGGCTGCCCCTGCGTAGCGGTCTGGGTGGTGCTCCACTGGGTCTGCGTCTGCCCCGGGAGACCGACGAA G 3′ (SEQ ID No. 39). The sequence of theantisense linker is 5′CTTCGTCGGTCTCCCGGGGCAGACGCAGACCCAGTGGAGCACCACCCAGACCGCTACGCAGGGGCAGCCGGATGCCG 3′ (SEQ ID No. 40). After annealing thephosphorylated linkers together in 0.1 M NaCl-10 mM Tris, pH 7.4 theywere ligated into unique Cla I and Sma I sites in Hu-Asp2 in the vectorpTAC. For inducible expression using induction with isopropylb-D-thiogalactopyranoside (IPTG), bacterial cultures were grown in LBbroth in the presence of ampicillin at 100 ug/ml, and induced in logphase growth at an OD600 of 0.6-1.0 with 1 mM IPTG for 4 hour at 37° C.The cell pellet was harvested by centrifugation.

To create a vector in which the leader sequences can be removed bylimited proteolysis with caspase 8 such that this liberates a Hu-Asp2polypeptide beginning with the N-terminal sequence GSFV (SEQ ID Nos. 27and 28), the following procedure was followed. Two phosphorylatedoligonucleotides containing the caspase 8 cleavage site IETD, #571=5′GATCGATGACTATCTCTGACTCTCCGCTGGACTCTGGTATCGAAACCGACG (SEQ ID No. 41) and#572=GATCCGTCGGTTTCGATACCAGAGTCCAGCGGAGAGTCAGAGATAGTCAT C (SEQ ID No.42) were annealed and ligated into pET23a+ that had been opened withBamHI. After transformation into JM109, the purified vector DNA wasrecovered and orientation of the insert was confirmed by DNA sequenceanalysis.

The following oligonucleotides were used for amplification of theselected Hu-Asp2(a) sequence: #573=5′AAGGATCCTTTGTGGAGATGGTGGACAACCTG,(SEQ ID No. 43) #554=GAAAGCTTTCATGACTCATCTGTCTGTGGAATGTTG (SEQ ID No.44) which placed BamHI and HindIII sites flanking the 5′ and 3′ ends ofthe insert, respectively. The Hu-Asp2(a) sequence was amplified from thefull length Hu-Asp2(a) cDNA cloned into pcDNA3.1 using the Advantage-GCcDNA PCR [Clontech] following the manufacturer's supplied protocol usingannealing & extension at 68° C. in a two-step PCR cycle for 25 cycles.The insert and vector were cut with BamHI and HindIII, purified byelectrophoresis through an agarose gel, then ligated using the Rapid DNALigation kit [Boerhinger Mannheim]. The ligation reaction was used totransform the E. coli strain JM109 [Promega] and colonies were pickedfor the purification of plasmid (Qiagen,Qiaprep minispin) and DNAsequence analysis. For inducible expression using induction withisopropyl b-D-thiogalactopyranoside (IPTG), the expression vector wastransferred into E. coli strain BL21 (Statagene). Bacterial cultureswere grown in LB broth in the presence of ampicillin at 100 ug/ml, andinduced in log phase growth at an OD600 of 0.6-1.0 with 1 mM IPTG for 4hour at 37° C. The cell pellet was harvested by centrifugation.

To assist purification, a 6-His tag can be introduced into any of theabove constructs following the T7 leader by opening the construct at theBamHI site and then ligating in the annealed, phosphorylatedoligonucleotides containing the six histidine sequence#565=GATCGCATCATCACCATCACCATG (SEQ ID No. 45),#566=GATCCATGGTGATGGTGATGATGC (SEQ ID No. 46). The 5′ overhang for eachset of oligonucleotides was designed such that it allowed ligation intothe BamHI site but not subsequent digestion with BamHI.

Preparation of Bacterial Pellet

36.34 g of bacterial pellet representing 10.8 L of growth was dispersedinto a total volume of 200 ml using a 20 mm tissue homogenizer probe at3000 to 5000 rpm in 2M KCl, 0.1M Tris, 0.05M EDTA, 1 mM DTT. Theconductivity adjusted to about 193 mMhos with water. After the pelletwas dispersed, an additional amount of the KCl solution was added,bringing the total volume to 500 ml. This suspension was homogenizedfurther for about 3 minutes at 5000 rpm using the same probe. Themixture was then passed through a Rannie high-pressure homogenizer at10,000 psi.

In all cases, the pellet material was carried forward, while the solublefraction was discarded. The resultant solution was centrifuged in a GSArotor for 1 hour at 12,500 rpm. The pellet was resuspended in the samesolution (without the DTT) using the same tissue homogenizer probe at2,000 rpm. After homogenizing for 5 minutes at 3000 rpm, the volume wasadjusted to 500 ml with the same solution, and spun for 1 hour at 12,500rpm. The pellet was then resuspended as before, but this time the finalvolume was adjusted to 1.5 L with the same solution prior tohomogenizing for 5 minutes. After centrifuging at the same speed for 30minutes, this procedure was repeated. The pellet was then resuspendedinto about 150 ml of cold water, pooling the pellets from the sixcentrifuge tubes used in the GSA rotor. The pellet has homogenized for 5minutes at 3,000 rpm, volume adjusted to 250 ml with cold water, thenspun for 30 minutes. Weight of the resultant pellet was 17.75 g.

Summary: Lysis of bacterial pellet in KCI solution, followed bycentrifugation in a GSA-rotor was used to initially prepare the pellet.The same solution was then used an additional three times forresuspension/homogenization. A final water wash/homogenization was thenperformed to remove excess KCl and EDTA.

Solublization of Recombinant Hu-Asp2(a)

A ratio of 9-10 ml/gram of pellet was utilized for solubilizing therHuAsp2L from the pellet previously described. 17.75 g of pellet wasthawed, and 150 ml of 8M guanidine HCl, 5 mM βME, 0.1% DEA, was added.3M Tris was used to titrate the pH to 8.6. The pellet was initiallyresuspended into the guanidine solution using a 20 mm tissue homogenizerprobe at 1000 rpm. The mixture was then stirred at 4° C. for 1 hourprior to centrifugation at 12,500 rpm for 1 hour in GSA rotor. Theresultant supernatant was then centrifuged for 30 minutes at 40,000×g inan SS-34 rotor. The final supernatant was then stored at −20° C., exceptfor 50 ml.

Immobilized Nickel Affinity Chromatography of Solubilized RecombinantHu-Asp2(a)

The following solutions were utilized:

A) 6M Guanidine HCl, 0.1M NaP, pH 8.0, 0.01M Tris, 5 mM βME, 0.5 mMImidazole

A′) 6M Urea, 20 mM NaP, pH 6.80, 50 mM NaCl

B′) 6M Urea, 20 mM NaP, pH 6.20, 50 mM NaCl, 12 mM Imidazole

C′) 6M Urea, 20 mM NaP, pH 6.80, 50 mM NaCl, 300 mM Imidazole

Note: Buffers A′ and C′ were mixed at the appropriate ratios to giveintermediate concentrations of Imidazole.

The 50 ml of solubilized material was combined with 50 ml of buffer Aprior to adding to 100-125 ml Qiagen Ni-NTA SuperFlow (pre-equilibratedwith buffer A) in a 5×10 cm Bio-Rad econo column. This was shaken gentlyovernight at 4° C. in the cold room.

Chromatography Steps

Drained the resultant flow through.

Washed with 50 ml buffer A (collecting into flow through fraction)

Washed with 250 ml buffer A (wash 1)

Washed with 250 ml buffer A (wash 2)

Washed with 250 ml buffer A′

Washed with 250 ml buffer B′

Washed with 250 ml buffer A′

Eluted with 250 ml 75 mM Imidazole

Eluted with 250 ml 150 mM Imidazole (150-1)

Eluted with 250 ml 150 mM Imidazole (150-2)

Eluted with 250 ml 300 mM Imidazole (300-1)

Eluted with 250 ml 300 mM Imidazole (300-2)

Eluted with 250 ml 300 mM Imidazole (300-3)

Chromatography Results

The Hu-Asp(a) eluted at 75 mM Imidazole through 300 mM Imidazole. The 75mM fraction, as well as the first 150 mM Imidazole (150-1) fractioncontained contaminating proteins as visualized on Coomassie Blue stainedgels. Therefore, fractions 150-2 and 300-1 will be utilized forrefolding experiments since they contained the greatest amount ofprotein as visualized on a Coomassie Blue stained gel.

Refolding Experiments of Recombinant Hu-Asp2(a)

Experiment 1

Forty ml of 150-2 was spiked with 1M DTT, 3M Tris, pH 7.4 and DEA to afinal concentration of 6 mM, 50 mM, and 0.1% respectively. This wasdiluted suddenly (while stirring) with 200 ml of (4° C.) cold 20 mM NaP,pH 6.8, 150 mM NaCl. This dilution gave a final Urea concentration of1M. This solution remained clear, even if allowed to set open to the airat room temperature (RT) or at 4° C. After setting open to the air for4-5 hours at 4° C., this solution was then dialyzed overnight against 20mM NaP, pH 7.4, 150 mM NaCl, 20% glycerol. This method effectivelyremoves the urea in the solution without precipitation of the protein.

Experiment 2

Some of the 150-2 eluate was concentrated 2× on an Amicon Centriprep,10,000 MWCO, then treated as in Experiment 1. This material also stayedin solution, with no visible precipitation.

Experiment 3

89 ml of the 150-2 eluate was spiked with 1M DTT, 3M Tris, pH 7.4 andDEA to a final concentration of 6 mM, 50 mM, and 0.1% respectively. Thiswas diluted suddenly (while stirring) with 445 ml of (4° C.) cold 20 mMNaP, pH 6.8, 150 mM NaCl. This solution appeared clear, with no apparentprecipitation. The solution was removed to RT and stirred for 10 minutesprior to adding MEA to a final concentration of 0.1 MM. This was stirredslowly at RT for 1 hour. Cystamine and CuSO₄ were then added to finalconcentrations of 1 mM and 10 μM respectively. The solution was stirredslowly at RT for 10 minutes prior to being moved to the 4° C. cold roomand shaken slowly overnight, open to the air.

The following day, the solution (still clear, with no apparentprecipitation) was centrifuged at 100,000×g for 1 hour. Supernatantsfrom multiple runs were pooled, and the bulk of the stabilized proteinwas dialyzed against 20 mM NaP, pH 7.4, 150 mM NaCl, 20% glycerol. Afterdialysis, the material was stored at −20° C.

Some (about 10 ml) of the protein solution (still in 1M Urea) was savedback for biochemical analyses, and frozen at −20° C. for storage.

EXAMPLE 10 Expression of Hu-Asp2 and Derivatives in Insect Cells

Any materials (vectors, host cells, etc.) and methods that are useful toexpress Hu-Asp2(a) should in principle be equally effective forexpression of Hu-Asp2(b).

Expression by Baculovirus Infection

The coding sequence of Hu-Asp2(a) and Hu-ASp2(b) and several derivativeswere engineered for expression in insect cells using the PCR. For thefull-length sequence, a 5′-sense oligonucleotide primer that modifiedthe translation initiation site to fit the Kozak consensus sequence waspaired with a 3′-antisense primer that contains the natural translationtermination codon in the Hu-Asp2 sequence. PCR amplification of thepcDNA3.1(hygro)/Hu-Asp2(a) template was used to prepare two derivativesof Hu-Asp2(a) or Hu-Asp(b) that delete the C-terminal transmembranedomain (SEQ ID Nos. 29-30 and 50-51, respectively) or delete thetransmembrane domain and introduce a hexa-histidine tag at theC-terminus (SEQ ID Nos. 31-32 and 52-53) respectively, were alsoengineered using PCR. The same 5′-sense oligonucleotide primer describedabove was paired with either a 3′-antisense primer that (1) introduced atranslation termination codon after codon 453 (SEQ ID No. 3) or (2)incorporated a hexa-histidine tag followed by a translation terminationcodon in the PCR using pcDNA3.1 (hygro)/Hu-Asp-2(a) as the template. Inall cases, the PCR reactions were performed amplified for 15 cyclesusing PwoI DNA polymerase (Boehringer-Mannheim) as outlined by thesupplier. The reaction products were digested to completion with BamHIand NotI and ligated to BamHI and NotI digested baculovirus transfervector pVL1393 (Invitrogen). A portion of the ligations was used totransform competent E. coli DH5_cells followed by antibiotic selectionon LB-Amp. Plasmid DNA was prepared by standard alkaline lysis andbanding in CsCl to yield the baculovirus transfer vectorspVL1393/Asp2(a), pVL1393/Asp2(a)ΔTM and pVL1393/Asp2(a)ΔTM(His)₆.Creation of recombinant baculoviruses and infection of sf9 insect cellswas performed using standard methods.

Expression by Transfection

Transient and stable expression of Hu-Asp2(a)ΔTM and Hu-Asp2(a)ΔTM(His)₆in High 5 insect cells was performed using the insect expression vectorpIZ/V5-His. The DNA inserts from the expression plasmids vectorspVL1393/Asp2(a), pVL1393/Asp2(a)ΔTM and pVL1393/Asp2(a)ΔTM(His)₆ wereexcised by double digestion with BamHI and NotI and subcloned into BamHIand NotI digested pIZ/V5-His using standard methods. The resultingexpression plasmids, referred to as pIZ/Hu-Asp2ΔTM andpIZ/Hu-Asp2Δ2TM(His)₆, were prepared as described above.

For transfection, High 5 insect cells were cultured in High Five serumfree medium supplemented with 10 μg/ml gentamycin at 27° C. in sealedflasks. Transfections were performed using High five cells, High fiveserum free media supplemented with 10 μg/ml gentamycin, and InsectinPlusliposomes (Invitrogen, Carlsbad, Calif.) using standard methods.

For large scale transient transfections, 1.2×10⁷ high five cells wereplated in a 150 mm tissue culture dish and allowed to attach at roomtemperature for 15-30 minutes. During the attachment time theDNA/liposome mixture was prepared by mixing 6 ml of serum free media, 60μg Hu-Asp2(a)ΔTM/pIZ (+/−His) DNA and 120 μl of Insectin Plus andincubating at room temperature for 15 minutes. The plating media wasremoved from the dish of cells and replaced with the DNA/liposomemixture for 4 hours at room temperature with constant rocking at 2 rpm.An additional 6 ml of media was added to the dish prior to incubationfor 4 days at 27° C. in a humid incubator. Four days post transfectionthe media was harvested, clarified by centrifugation at 500×g, assayedfor Hu-Asp2(a) expression by Western blotting. For stable expression,the cells were treated with 50 μg/ml Zeocin and the surviving pool usedto prepared clonal cells by limiting dilution followed by analysis ofthe expression level as noted above.

Purification of Hu-Asp2(a)ΔTM and Hu-Asp2(a)ΔTM(His)₆

Removal of the transmembrane segment from Hu-Asp2(a) resulted in thesecretion of the polypeptide into the culture medium. Following proteinproduction by either baculovirus infection or transfection, theconditioned medium was harvested, clarified by centrifugation, anddialyzed against Tris-HCl (pH 8.0). This material was then purified bysuccessive chromatography by anion exchange (Tris-HCl, pH 8.0) followedby cation exchange chromatography (Acetate buffer at pH 4.5) using NaClgradients. The elution profile was monitored by (1) Western blotanalysis and (2) by activity assay using the peptide substrate describedin Example 12. For the Hu-Asp2(a)ΔTM(His)₆, the conditioned medium wasdialyzed against Tris buffer (pH 8.0) and purified by sequentialchromatography on IMAC resin followed by anion exchange chromatography.

Amino-terminal sequence analysis of the purified Hu-Asp2(a)ΔTM(His)₆protein revealed that the signal peptide had been cleaved [TQHGIRLPLR,corresponding to SEQ ID NO: 32, residues 22-3].

EXAMPLE 11 Expression of Hu-Asp2(a) and Hu-Asp(b) in CHO Cells

The materials (vectors, host cells, etc.) and methods described hereinfor expression of Hu-Asp2(a) are intended to be equally applicable forexpression of Hu-Asp2(b).

Heterologous Expression of Hu-Asp-2(a) in CHO-K1 Cells

The entire coding sequence of Hu-Asp2(a) was cloned into the mammalianexpression vector pcDNA3. 1 (+)Hygro (Invitrogen, Carlsbad, Calif.)which contains the CMV immediate early promoter and bGH polyadenylationsignal to drive over expression. The expression plasmid, pcDNA3.1(+)Hygro/Hu-Asp2(a), was prepared by alkaline lysis and banding in CsCland completely sequenced on both strands to verify the integrity of thecoding sequence.

Wild-type Chinese hamster ovary cells (CHO-K1) were obtained from theATCC. The cells were maintained in monolayer cultures in α-MEMcontaining 10% FCS at 37° C. in 5% CO₂. Two 100 mm dishes of CHO-K1cells (60% confluent) were transfected with pcDNA3.1(+)/Hygro alone(mock) or pcDNA3.1(+)Hygro/Hu-Asp2(a) or pcDNA3.1(+)Hygro/Hu-Asp2(b)using the cationic liposome DOTAP as recommended by the supplier (Roche,Indianapolis, Ind.). The cells were treated with the plasmidDNA/liposome mixtures for 15 hours and then the medium replaced withgrowth medium containing 500 Units/ml hygromycin B. In the case ofpcDNA3.1(+)Hygro/Hu-Asp2(a) or (b) transfected CHO-K1 cells, individualhygromycin B-resistant cells were cloned by limiting dilution. Followingclonal expansion of the individual cell lines, expression of Hu-Asp2(a)or Hu-Asp2(b) protein was assessed by Western blot analysis using apolyclonal rabbit antiserum raised against recombinant Hu-Asp2 preparedby expression in E. coli. Near confluent dishes of each cell line wereharvested by scraping into PBS and the cells recovered bycentrifugation. The cell pellets were resuspended in cold lysis buffer(25 mM Tris-HCl (pH 8.0)/5 mM EDTA) containing protease inhibitors andthe cells lysed by sonication. The soluble and membrane fractions wereseparated by centrifugation (105,000×g, 60 min) and normalized amountsof protein from each fraction were then separated by SDS-PAGE. Followingelectrotransfer of the separated polypeptides to PVDF membranes,Hu-Asp-2(a) or Hu-Asp2(b) protein was detected using rabbit anti-Hu-Asp2antiserum (1/1000 dilution) and the antibody-antigen complexes werevisualized using alkaline phosphatase conjugated goat anti-rabbitantibodies (1/2500). A specific immunoreactive protein with an apparentMr value of 65 kDa was detected in pcDNA3.1(+)Hygro/Hu-Asp2 transfectedcells and not mock-transfected cells. Also, the Hu-Asp2 polypeptide wasonly detected in the membrane fraction, consistent with the presence ofa signal peptide and single transmembrane domain in the predictedsequence. Based on this analysis, clone #5 had the highest expressionlevel of Hu-Asp2(a) protein and this production cell lines was scaled upto provide material for purification.

Purification of Recombinant Hu-Asp-2(a) from CHO-K1/Hu-Asp2 Clone #5

In a typical purification, clone #5 cell pellets derived from 20 150 mmdishes of confluent cells, were used as the starting material. The cellpellets were resuspended in 50 ml cold lysis buffer as described above.The cells were lysed by polytron homogenization (2×20 sec) and thelysate centrifuged at 338,000×g for 20 minutes. The membrane pellet wasthen resuspended in 20 ml of cold lysis buffer containing 50 mMβ-octylglucoside followed by rocking at 4° C. for 1 hour. The detergentextract was clarified by centrifugation at 338,000×g for 20 minutes andthe supernatant taken for further analysis.

The β-octylglucoside extract was applied to a Mono Q anion exchangecolumn that was previously equilibrated with 25 mM Tris-HCl (pH 8.0)/50mM β-octylglucoside. Following sample application, the column was elutedwith a linear gradient of increasing NaCl concentration (0-1.0 M over 30minutes) and individual fractions assayed by Western blot analysis andfor β-secretase activity (see below). Fractions containing bothHu-Asp-2(a) immunoreactivity and β-secretase activity were pooled anddialyzed against 25 mM NaOAc (pH 4.5)/50 mM β-octylglucoside. Followingdialysis, precipitated material was removed by centrifugation and thesoluble material chromatographed on a MonoS cation exchange column thatwas previously equilibrated in 25 mM NaOAc (pH 4.5)/50 mMβ-octylglucoside. The column was eluted using a linear gradient ofincreasing NaCl concentration (0-1.0 M over 30 minutes) and individualfractions assayed by Western blot analysis and for β-secretase activity.Fractions containing both Hu-Asp2 immunoreactivity and β-secretaseactivity were combined and determined to be >95% pure bySDS-PAGE/Coomassie Blue staining.

The same methods were used to express and purify Hu-Asp2(b).

EXAMPLE 12 Assay of Hu-Asp2 β-secretase Activity Using PeptideSubstrates

β-secretase Assay

Recombinant human Asp2(a) prepared in CHO cells and purified asdescribed in Example 11 was used to assay Asp2(a) proteolytic activitydirectly. Activity assays for Asp2(a) were performed using syntheticpeptide substrates containing either the wild-type APP β-secretase site(SEVKM↓DAEFR SEQ ID NO:64), the Swedish KM→NL mutation (SEVNL↓DAEFR SEQID NO:63), or the βP40 and 42 γ-secretase sites (RRGGVVIA↓TVIVGER SEQ IDNO:65). Reactions were performed in 50 nM2-[N-morpholino]ethane-sulfonate (“Na-MES,” pH 5.5) containing 1%β-octylglucoside, 70 mM peptide substrate, and recombinant Asp2(a) (1-5μg protein) for various times at 37° C. The reaction products werequantified by RP-HPLC using a linear gradient from 0-70 B over 30minutes (A=0.1% TFA in water, B=0.1%TFA/10% water/90%AcCN). The elutionprofile was monitored by absorbance at 214 nm. In preliminaryexperiments, the two product peaks which eluted before the intactpeptide substrate, were confirmed to have the sequence DAEFR and SEVNLusing both Edman sequencing and MADLI-TOF mass spectrometry. Percenthydrolysis of the peptide substrate was calculated by comparing theintegrated peak areas for the two product peptides and the startingmaterial derived from the absorbance at 214 nm. The sequence ofcleavage/hydrolysis products was confirmed using Edman sequencing andMADLI-TOF mass spectrometry.

The behavior of purified Asp2(a) in the proteolysis assays wasconsistent with the prior anti-sense studies which indicated thatAsp2(a) possesses β-secretase activity. Maximal proteolysis was seenwith the Swedigh β-secretase peptide, which, after 6 hours, was about10-fold higher than wild type APP.

The specificity of the protease cleavage reaction was determined byperforming the β-secretase assay in the presence of 8 μM pepstatin A andthe presence of a cocktail of protease inhibitors (10 μM leupeptin, 10μM E64, and 5 mM EDTA). Proteolytic activity was insensitive to both thepepstatin and the cocktail, which are inhibitors of cathepsin D (andother aspartyl proteases), serine proteases, cysteinyl proteases, andmetalloproteases, respectively.

Hu-Asp2(b) when similarly expressed in CHO cells and purified usingidentical conditions for extraction with β-octylglucoside and sequentialchromatography over Mono Q and Mono S also cleaves the Swedishβ-secretase peptide in proteolysis assays using identical assayconditions.

Collectively, this data establishes that both forms of Asp2 (Hu-Asp2(a)and Hu-Asp2(b)) act directly in cell-free assays to cleave synthetic APPpeptides at the β-secretase site, and that the rate of cleavage isgreatly increased by the Swedish KM→NL mutation that is associated withAlzheimer's disease.

An alternative β-secretase assay utilizes internally quenchedfluorescent substrates to monitor enzyme activity using fluorescencespectroscopy in a single sample or multiwell format. Each reactioncontained 50 mM Na-MES (pH 5.5), peptide substrate MCA-EVKMDAEF[K-DNP](SEQ ID NO:71) (BioSource International) (50 μM) and purified Hu-Asp-2enzyme. These components were equilibrated to 37° C. for various timesand the reaction initiated by addition of substrate. Excitation wasperformed at 330 nm and the reaction kinetics were monitored bymeasuring the fluorescence emission at 390 nm. To detect compounds thatmodulate Hu-Asp-2 activity, the test compounds were added during thepreincubation phase of the reaction and the kinetics of the reactionmonitored as described above. Activators are scored as compounds thatincrease the rate of appearance of fluorescence while inhibitorsdecrease the rate of appearance of fluorescence.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the invention. The entire disclosure of all publications citedherein are hereby incorporated by reference.

73 1 1804 DNA Homo sapiens 1 atgggcgcac tggcccgggc gctgctgctg cctctgctggcccagtggct cctgcgcgcc 60 gccccggagc tggcccccgc gcccttcacg ctgcccctccgggtggccgc ggccacgaac 120 cgcgtagttg cgcccacccc gggacccggg acccctgccgagcgccacgc cgacggcttg 180 gcgctcgccc tggagcctgc cctggcgtcc cccgcgggcgccgccaactt cttggccatg 240 gtagacaacc tgcaggggga ctctggccgc ggctactacctggagatgct gatcgggacc 300 cccccgcaga agctacagat tctcgttgac actggaagcagtaactttgc cgtggcagga 360 accccgcact cctacataga cacgtacttt gacacagagaggtctagcac ataccgctcc 420 aagggctttg acgtcacagt gaagtacaca caaggaagctggacgggctt cgttggggaa 480 gacctcgtca ccatccccaa aggcttcaat acttcttttcttgtcaacat tgccactatt 540 tttgaatcag agaatttctt tttgcctggg attaaatggaatggaatact tggcctagct 600 tatgccacac ttgccaagcc atcaagttct ctggagaccttcttcgactc cctggtgaca 660 caagcaaaca tccccaacgt tttctccatg cagatgtgtggagccggctt gcccgttgct 720 ggatctggga ccaacggagg tagtcttgtc ttgggtggaattgaaccaag tttgtataaa 780 ggagacatct ggtatacccc tattaaggaa gagtggtactaccagataga aattctgaaa 840 ttggaaattg gaggccaaag ccttaatctg gactgcagagagtataacgc agacaaggcc 900 atcgtggaca gtggcaccac gctgctgcgc ctgccccagaaggtgtttga tgcggtggtg 960 gaagctgtgg cccgcgcatc tctgattcca gaattctctgatggtttctg gactgggtcc 1020 cagctggcgt gctggacgaa ttcggaaaca ccttggtcttacttccctaa aatctccatc 1080 tacctgagag atgagaactc cagcaggtca ttccgtatcacaatcctgcc tcagctttac 1140 attcagccca tgatgggggc cggcctgaat tatgaatgttaccgattcgg catttcccca 1200 tccacaaatg cgctggtgat cggtgccacg gtgatggagggcttctacgt catcttcgac 1260 agagcccaga agagggtggg cttcgcagcg agcccctgtgcagaaattgc aggtgctgca 1320 gtgtctgaaa tttccgggcc tttctcaaca gaggatgtagccagcaactg tgtccccgct 1380 cagtctttga gcgagcccat tttgtggatt gtgtcctatgcgctcatgag cgtctgtgga 1440 gccatcctcc ttgtcttaat cgtcctgctg ctgctgccgttccggtgtca gcgtcgcccc 1500 cgtgaccctg aggtcgtcaa tgatgagtcc tctctggtcagacatcgctg gaaatgaata 1560 gccaggcctg acctcaagca accatgaact cagctattaagaaaatcaca tttccagggc 1620 agcagccggg atcgatggtg gcgctttctc ctgtgcccacccgtcttcaa tctctgttct 1680 gctcccagat gccttctaga ttcactgtct tttgattcttgattttcaag ctttcaaatc 1740 ctccctactt ccaagaaaaa taattaaaaa aaaaacttcattctaaacca aaaaaaaaaa 1800 aaaa 1804 2 518 PRT Homo sapiens 2 Met GlyAla Leu Ala Arg Ala Leu Leu Leu Pro Leu Leu Ala Gln Trp 1 5 10 15 LeuLeu Arg Ala Ala Pro Glu Leu Ala Pro Ala Pro Phe Thr Leu Pro 20 25 30 LeuArg Val Ala Ala Ala Thr Asn Arg Val Val Ala Pro Thr Pro Gly 35 40 45 ProGly Thr Pro Ala Glu Arg His Ala Asp Gly Leu Ala Leu Ala Leu 50 55 60 GluPro Ala Leu Ala Ser Pro Ala Gly Ala Ala Asn Phe Leu Ala Met 65 70 75 80Val Asp Asn Leu Gln Gly Asp Ser Gly Arg Gly Tyr Tyr Leu Glu Met 85 90 95Leu Ile Gly Thr Pro Pro Gln Lys Leu Gln Ile Leu Val Asp Thr Gly 100 105110 Ser Ser Asn Phe Ala Val Ala Gly Thr Pro His Ser Tyr Ile Asp Thr 115120 125 Tyr Phe Asp Thr Glu Arg Ser Ser Thr Tyr Arg Ser Lys Gly Phe Asp130 135 140 Val Thr Val Lys Tyr Thr Gln Gly Ser Trp Thr Gly Phe Val GlyGlu 145 150 155 160 Asp Leu Val Thr Ile Pro Lys Gly Phe Asn Thr Ser PheLeu Val Asn 165 170 175 Ile Ala Thr Ile Phe Glu Ser Glu Asn Phe Phe LeuPro Gly Ile Lys 180 185 190 Trp Asn Gly Ile Leu Gly Leu Ala Tyr Ala ThrLeu Ala Lys Pro Ser 195 200 205 Ser Ser Leu Glu Thr Phe Phe Asp Ser LeuVal Thr Gln Ala Asn Ile 210 215 220 Pro Asn Val Phe Ser Met Gln Met CysGly Ala Gly Leu Pro Val Ala 225 230 235 240 Gly Ser Gly Thr Asn Gly GlySer Leu Val Leu Gly Gly Ile Glu Pro 245 250 255 Ser Leu Tyr Lys Gly AspIle Trp Tyr Thr Pro Ile Lys Glu Glu Trp 260 265 270 Tyr Tyr Gln Ile GluIle Leu Lys Leu Glu Ile Gly Gly Gln Ser Leu 275 280 285 Asn Leu Asp CysArg Glu Tyr Asn Ala Asp Lys Ala Ile Val Asp Ser 290 295 300 Gly Thr ThrLeu Leu Arg Leu Pro Gln Lys Val Phe Asp Ala Val Val 305 310 315 320 GluAla Val Ala Arg Ala Ser Leu Ile Pro Glu Phe Ser Asp Gly Phe 325 330 335Trp Thr Gly Ser Gln Leu Ala Cys Trp Thr Asn Ser Glu Thr Pro Trp 340 345350 Ser Tyr Phe Pro Lys Ile Ser Ile Tyr Leu Arg Asp Glu Asn Ser Ser 355360 365 Arg Ser Phe Arg Ile Thr Ile Leu Pro Gln Leu Tyr Ile Gln Pro Met370 375 380 Met Gly Ala Gly Leu Asn Tyr Glu Cys Tyr Arg Phe Gly Ile SerPro 385 390 395 400 Ser Thr Asn Ala Leu Val Ile Gly Ala Thr Val Met GluGly Phe Tyr 405 410 415 Val Ile Phe Asp Arg Ala Gln Lys Arg Val Gly PheAla Ala Ser Pro 420 425 430 Cys Ala Glu Ile Ala Gly Ala Ala Val Ser GluIle Ser Gly Pro Phe 435 440 445 Ser Thr Glu Asp Val Ala Ser Asn Cys ValPro Ala Gln Ser Leu Ser 450 455 460 Glu Pro Ile Leu Trp Ile Val Ser TyrAla Leu Met Ser Val Cys Gly 465 470 475 480 Ala Ile Leu Leu Val Leu IleVal Leu Leu Leu Leu Pro Phe Arg Cys 485 490 495 Gln Arg Arg Pro Arg AspPro Glu Val Val Asn Asp Glu Ser Ser Leu 500 505 510 Val Arg His Arg TrpLys 515 3 2070 DNA Homo sapiens 3 atggcccaag ccctgccctg gctcctgctgtggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccg gctgcccctgcgcagcggcc tggggggcgc ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagagcccgaggagc ccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaagtcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatcctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcatcgctactacc agaggcagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtgccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag catcccccatggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcaga caagttcttcatcaacggct ccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggcctgacgactccc tggagccttt ctttgactct 600 ctggtaaagc agacccacgt tcccaacctcttctccctgc agctttgtgg tgctggcttc 660 cccctcaacc agtctgaagt gctggcctctgtcggaggga gcatgatcat tggaggtatc 720 gaccactcgc tgtacacagg cagtctctggtatacaccca tccggcggga gtggtattat 780 gaggtcatca ttgtgcgggt ggagatcaatggacaggatc tgaaaatgga ctgcaaggag 840 tacaactatg acaagagcat tgtggacagtggcaccacca accttcgttt gcccaagaaa 900 gtgtttgaag ctgcagtcaa atccatcaaggcagcctcct ccacggagaa gttccctgat 960 ggtttctggc taggagagca gctggtgtgctggcaagcag gcaccacccc ttggaacatt 1020 ttcccagtca tctcactcta cctaatgggtgaggttacca accagtcctt ccgcatcacc 1080 atccttccgc agcaatacct gcggccagtggaagatgtgg ccacgtccca agacgactgt 1140 tacaagtttg ccatctcaca gtcatccacgggcactgtta tgggagctgt tatcatggag 1200 ggcttctacg ttgtctttga tcgggcccgaaaacgaattg gctttgctgt cagcgcttgc 1260 catgtgcacg atgagttcag gacggcagcggtggaaggcc cttttgtcac cttggacatg 1320 gaagactgtg gctacaacat tccacagacagatgagtcaa ccctcatgac catagcctat 1380 gtcatggctg ccatctgcgc cctcttcatgctgccactct gcctcatggt gtgtcagtgg 1440 cgctgcctcc gctgcctgcg ccagcagcatgatgactttg ctgatgacat ctccctgctg 1500 aagtgaggag gcccatgggc agaagatagagattcccctg gaccacacct ccgtggttca 1560 ctttggtcac aagtaggaga cacagatggcacctgtggcc agagcacctc aggaccctcc 1620 ccacccacca aatgcctctg ccttgatggagaaggaaaag gctggcaagg tgggttccag 1680 ggactgtacc tgtaggaaac agaaaagagaagaaagaagc actctgctgg cgggaatact 1740 cttggtcacc tcaaatttaa gtcgggaaattctgctgctt gaaacttcag ccctgaacct 1800 ttgtccacca ttcctttaaa ttctccaacccaaagtattc ttcttttctt agtttcagaa 1860 gtactggcat cacacgcagg ttaccttggcgtgtgtccct gtggtaccct ggcagagaag 1920 agaccaagct tgtttccctg ctggccaaagtcagtaggag aggatgcaca gtttgctatt 1980 tgctttagag acagggactg tataaacaagcctaacattg gtgcaaagat tgcctcttga 2040 attaaaaaaa aaaaaaaaaa aaaaaaaaaa2070 4 501 PRT Homo sapiens 4 Met Ala Gln Ala Leu Pro Trp Leu Leu LeuTrp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr Gln His GlyIle Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Gly Gly Ala Pro Leu Gly LeuArg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Pro Glu Glu Pro Gly Arg ArgGly Ser Phe Val Glu Met Val 50 55 60 Asp Asn Leu Arg Gly Lys Ser Gly GlnGly Tyr Tyr Val Glu Met Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr LeuAsn Ile Leu Val Asp Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala AlaPro His Pro Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser SerThr Tyr Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr GlnGly Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile ProHis Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala IleThr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 Glu GlyIle Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp 180 185 190 SerLeu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr His Val Pro 195 200 205Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln 210 215220 Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile 225230 235 240 Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile ArgArg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile AsnGly Gln 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp LysSer Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys LysVal Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser Ser ThrGlu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly Glu Gln Leu ValCys Trp Gln Ala Gly Thr Thr 325 330 335 Pro Trp Asn Ile Phe Pro Val IleSer Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr Asn Gln Ser Phe Arg IleThr Ile Leu Pro Gln Gln Tyr Leu Arg 355 360 365 Pro Val Glu Asp Val AlaThr Ser Gln Asp Asp Cys Tyr Lys Phe Ala 370 375 380 Ile Ser Gln Ser SerThr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 400 Gly Phe TyrVal Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val SerAla Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430 GlyPro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445Gln Thr Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr Val Met Ala Ala 450 455460 Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu Met Val Cys Gln Trp 465470 475 480 Arg Cys Leu Arg Cys Leu Arg Gln Gln His Asp Asp Phe Ala AspAsp 485 490 495 Ile Ser Leu Leu Lys 500 5 1977 DNA Homo sapiens 5atggcccaag ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60ggcacccagc acggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 240gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 300gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540gggctggcct atgctgagat tgccaggctt tgtggtgctg gcttccccct caaccagtct 600gaagtgctgg cctctgtcgg agggagcatg atcattggag gtatcgacca ctcgctgtac 660acaggcagtc tctggtatac acccatccgg cgggagtggt attatgaggt gatcattgtg 720cgggtggaga tcaatggaca ggatctgaaa atggactgca aggagtacaa ctatgacaag 780agcattgtgg acagtggcac caccaacctt cgtttgccca agaaagtgtt tgaagctgca 840gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc ctgatggttt ctggctagga 900gagcagctgg tgtgctggca agcaggcacc accccttgga acattttccc agtcatctca 960ctctacctaa tgggtgaggt taccaaccag tccttccgca tcaccatcct tccgcagcaa 1020tacctgcggc cagtggaaga tgtggccacg tcccaagacg actgttacaa gtttgccatc 1080tcacagtcat ccacgggcac tgttatggga gctgttatca tggagggctt ctacgttgtc 1140tttgatcggg cccgaaaacg aattggcttt gctgtcagcg cttgccatgt gcacgatgag 1200ttcaggacgg cagcggtgga aggccctttt gtcaccttgg acatggaaga ctgtggctac 1260aacattccac agacagatga gtcaaccctc atgaccatag cctatgtcat ggctgccatc 1320tgcgccctct tcatgctgcc actctgcctc atggtgtgtc agtggcgctg cctccgctgc 1380ctgcgccagc agcatgatga ctttgctgat gacatctccc tgctgaagtg aggaggccca 1440tgggcagaag atagagattc ccctggacca cacctccgtg gttcactttg gtcacaagta 1500ggagacacag atggcacctg tggccagagc acctcaggac cctccccacc caccaaatgc 1560ctctgccttg atggagaagg aaaaggctgg caaggtgggt tccagggact gtacctgtag 1620gaaacagaaa agagaagaaa gaagcactct gctggcggga atactcttgg tcacctcaaa 1680tttaagtcgg gaaattctgc tgcttgaaac ttcagccctg aacctttgtc caccattcct 1740ttaaattctc caacccaaag tattcttctt ttcttagttt cagaagtact ggcatcacac 1800gcaggttacc ttggcgtgtg tccctgtggt accctggcag agaagagacc aagcttgttt 1860ccctgctggc caaagtcagt aggagaggat gcacagtttg ctatttgctt tagagacagg 1920gactgtataa acaagcctaa cattggtgca aagattgcct cttgaaaaaa aaaaaaa 1977 6476 PRT Homo sapiens 6 Met Ala Gln Ala Leu Pro Trp Leu Leu Leu Trp MetGly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr Gln His Gly Ile ArgLeu Pro Leu Arg Ser 20 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg LeuPro Arg Glu Thr Asp 35 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly SerPhe Val Glu Met Val 50 55 60 Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly TyrTyr Val Glu Met Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr Leu Asn IleLeu Val Asp Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro HisPro Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr TyrArg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln Gly LysTrp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile Pro His GlyPro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala Ile Thr GluSer Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 Glu Gly Ile LeuGly Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 180 185 190 Ala Gly PhePro Leu Asn Gln Ser Glu Val Leu Ala Ser Val Gly Gly 195 200 205 Ser MetIle Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu 210 215 220 TrpTyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val 225 230 235240 Arg Val Glu Ile Asn Gly Gln Asp Leu Lys Met Asp Cys Lys Glu Tyr 245250 255 Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu260 265 270 Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala AlaSer 275 280 285 Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu GlnLeu Val 290 295 300 Cys Trp Gln Ala Gly Thr Thr Pro Trp Asn Ile Phe ProVal Ile Ser 305 310 315 320 Leu Tyr Leu Met Gly Glu Val Thr Asn Gln SerPhe Arg Ile Thr Ile 325 330 335 Leu Pro Gln Gln Tyr Leu Arg Pro Val GluAsp Val Ala Thr Ser Gln 340 345 350 Asp Asp Cys Tyr Lys Phe Ala Ile SerGln Ser Ser Thr Gly Thr Val 355 360 365 Met Gly Ala Val Ile Met Glu GlyPhe Tyr Val Val Phe Asp Arg Ala 370 375 380 Arg Lys Arg Ile Gly Phe AlaVal Ser Ala Cys His Val His Asp Glu 385 390 395 400 Phe Arg Thr Ala AlaVal Glu Gly Pro Phe Val Thr Leu Asp Met Glu 405 410 415 Asp Cys Gly TyrAsn Ile Pro Gln Thr Asp Glu Ser Thr Leu Met Thr 420 425 430 Ile Ala TyrVal Met Ala Ala Ile Cys Ala Leu Phe Met Leu Pro Leu 435 440 445 Cys LeuMet Val Cys Gln Trp Arg Cys Leu Arg Cys Leu Arg Gln Gln 450 455 460 HisAsp Asp Phe Ala Asp Asp Ile Ser Leu Leu Lys 465 470 475 7 2043 DNA Musmusculus 7 atggccccag cgctgcactg gctcctgcta tgggtgggct cgggaatgctgcctgcccag 60 ggaacccatc tcggcatccg gctgcccctt cgcagcggcc tggcagggccacccctgggc 120 ctgaggctgc cccgggagac tgacgaggaa tcggaggagc ctggccggagaggcagcttt 180 gtggagatgg tggacaacct gaggggaaag tccggccagg gctactatgtggagatgacc 240 gtaggcagcc ccccacagac gctcaacatc ctggtggaca cgggcagtagtaactttgca 300 gtgggggctg ccccacaccc tttcctgcat cgctactacc agaggcagctgtccagcaca 360 tatcgagacc tccgaaaggg tgtgtatgtg ccctacaccc agggcaagtgggagggggaa 420 ctgggcaccg acctggtgag catccctcat ggccccaacg tcactgtgcgtgccaacatt 480 gctgccatca ctgaatcgga caagttcttc atcaatggtt ccaactgggagggcatccta 540 gggctggcct atgctgagat tgccaggccc gacgactctt tggagcccttctttgactcc 600 ctggtgaagc agacccacat tcccaacatc ttttccctgc agctctgtggcgctggcttc 660 cccctcaacc agaccgaggc actggcctcg gtgggaggga gcatgatcattggtggtatc 720 gaccactcgc tatacacggg cagtctctgg tacacaccca tccggcgggagtggtattat 780 gaagtgatca ttgtacgtgt ggaaatcaat ggtcaagatc tcaagatggactgcaaggag 840 tacaactacg acaagagcat tgtggacagt gggaccacca accttcgcttgcccaagaaa 900 gtatttgaag ctgccgtcaa gtccatcaag gcagcctcct cgacggagaagttcccggat 960 ggcttttggc taggggagca gctggtgtgc tggcaagcag gcacgaccccttggaacatt 1020 ttcccagtca tttcacttta cctcatgggt gaagtcacca atcagtccttccgcatcacc 1080 atccttcctc agcaatacct acggccggtg gaggacgtgg ccacgtcccaagacgactgt 1140 tacaagttcg ctgtctcaca gtcatccacg ggcactgtta tgggagccgtcatcatggaa 1200 ggtttctatg tcgtcttcga tcgagcccga aagcgaattg gctttgctgtcagcgcttgc 1260 catgtgcacg atgagttcag gacggcggca gtggaaggtc cgtttgttacggcagacatg 1320 gaagactgtg gctacaacat tccccagaca gatgagtcaa cacttatgaccatagcctat 1380 gtcatggcgg ccatctgcgc cctcttcatg ttgccactct gcctcatggtatgtcagtgg 1440 cgctgcctgc gttgcctgcg ccaccagcac gatgactttg ctgatgacatctccctgctc 1500 aagtaaggag gctcgtgggc agatgatgga gacgcccctg gaccacatctgggtggttcc 1560 ctttggtcac atgagttgga gctatggatg gtacctgtgg ccagagcacctcaggaccct 1620 caccaacctg ccaatgcttc tggcgtgaca gaacagagaa atcaggcaagctggattaca 1680 gggcttgcac ctgtaggaca caggagaggg aaggaagcag cgttctggtggcaggaatat 1740 ccttaggcac cacaaacttg agttggaaat tttgctgctt gaagcttcagccctgaccct 1800 ctgcccagca tcctttagag tctccaacct aaagtattct ttatgtccttccagaagtac 1860 tggcgtcata ctcaggctac ccggcatgtg tccctgtggt accctggcagagaaagggcc 1920 aatctcattc cctgctggcc aaagtcagca gaagaaggtg aagtttgccagttgctttag 1980 tgatagggac tgcagactca agcctacact ggtacaaaga ctgcgtcttgagataaacaa 2040 gaa 2043 8 501 PRT Mus musculus 8 Met Ala Pro Ala LeuHis Trp Leu Leu Leu Trp Val Gly Ser Gly Met 1 5 10 15 Leu Pro Ala GlnGly Thr His Leu Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Ala GlyPro Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Ser GluGlu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 50 55 60 Asp Asn Leu ArgGly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr 65 70 75 80 Val Gly SerPro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 85 90 95 Ser Asn PheAla Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 100 105 110 Tyr GlnArg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 115 120 125 TyrVal Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp165 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro AspAsp 180 185 190 Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr HisIle Pro 195 200 205 Asn Ile Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe ProLeu Asn Gln 210 215 220 Thr Glu Ala Leu Ala Ser Val Gly Gly Ser Met IleIle Gly Gly Ile 225 230 235 240 Asp His Ser Leu Tyr Thr Gly Ser Leu TrpTyr Thr Pro Ile Arg Arg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile ValArg Val Glu Ile Asn Gly Gln 260 265 270 Asp Leu Lys Met Asp Cys Lys GluTyr Asn Tyr Asp Lys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn LeuArg Leu Pro Lys Lys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile LysAla Ala Ser Ser Thr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp LeuGly Glu Gln Leu Val Cys Trp Gln Ala Gly Thr Thr 325 330 335 Pro Trp AsnIle Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr AsnGln Ser Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg 355 360 365 ProVal Glu Asp Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe Ala 370 375 380Val Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390395 400 Gly Phe Tyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala405 410 415 Val Ser Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala ValGlu 420 425 430 Gly Pro Phe Val Thr Ala Asp Met Glu Asp Cys Gly Tyr AsnIle Pro 435 440 445 Gln Thr Asp Glu Ser Thr Leu Met Thr Ile Ala Tyr ValMet Ala Ala 450 455 460 Ile Cys Ala Leu Phe Met Leu Pro Leu Cys Leu MetVal Cys Gln Trp 465 470 475 480 Arg Cys Leu Arg Cys Leu Arg His Gln HisAsp Asp Phe Ala Asp Asp 485 490 495 Ile Ser Leu Leu Lys 500 9 2088 DNAHomo sapiens 9 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggcgctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgttctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatcagggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtctaccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactggtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctgcttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttacaccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagacatgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattgacaagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtggattctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagactatgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggtggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaagaggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccaccaccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccctgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttccagaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagagaatgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttatccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagcagctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggccctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaatatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcatttcgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgacacacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcctgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaactattcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgctctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggagagttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaacacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccactcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaag tgaagatggatgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgcagaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcatagcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaag aaacagtaca catccattcatcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagatgcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag2088 10 695 PRT Homo sapiens 10 Met Leu Pro Gly Leu Ala Leu Leu Leu LeuAla Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly AsnAla Gly Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg LeuAsn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro SerGly Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr CysGln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu AlaAsn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys GlnCys Lys Thr His Pro His Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu ValGly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys LysPhe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu HisTrp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser ThrAsn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp LysPhe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 SerAsp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val GluGlu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr ThrSer Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu GluVal Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala ValAsp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala HisPhe Gln Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg GluArg Met Ser Gln Val Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg GlnAla Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile Gln HisPhe Gln Glu Lys Val Glu Ser Leu Glu 355 360 365 Gln Glu Ala Ala Asn GluArg Gln Gln Leu Val Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala MetLeu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile ThrAla Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn MetLeu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His 420 425 430 ThrLeu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455460 Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465470 475 480 Glu Glu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu GlnAsn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro ArgIle Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu ThrLys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser LeuAsp Asp Leu Gln 530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val ProAla Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg ProAla Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu ThrAsn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala GluPhe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His His Gln Lys Leu ValPhe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile GlyLeu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635 640 Ile Val IleThr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile 645 650 655 His HisGly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 HisLeu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685Phe Phe Glu Gln Met Gln Asn 690 695 11 2088 DNA Homo sapiens 11atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840gagtctgtgg aagaggtggt tcgagttcct acaacagcag ccagtacccc tgatgccgtt 900gacaagtatc tcgagacacc tggggatgag aatgaacatg cccatttcca gaaagccaaa 960gagaggcttg aggccaagca ccgagagaga atgtcccagg tcatgagaga atgggaagag 1020gcagaacgtc aagcaaagaa cttgcctaaa gctgataaga aggcagttat ccagcatttc 1080caggagaaag tggaatcttt ggaacaggaa gcagccaacg agagacagca gctggtggag 1140acacacatgg ccagagtgga agccatgctc aatgaccgcc gccgcctggc cctggagaac 1200tacatcaccg ctctgcaggc tgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260aagtatgtcc gcgcagaaca gaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320cgcatggtgg atcccaagaa agccgctcag atccggtccc aggttatgac acacctccgt 1380gtgatttatg agcgcatgaa tcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440gaggagattc aggatgaagt tgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500gtcttggcca acatgattag tgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560tctttgaccg aaacgaaaac caccgtggag ctccttcccg tgaatggaga gttcagcctg 1620gacgatctcc agccgtggca ttcttttggg gctgactctg tgccagccaa cacagaaaac 1680gaagttgagc ctgttgatgc ccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740tctgggttga caaatatcaa gacggaggag atctctgaag tgaatctgga tgcagaattc 1800cgacatgact caggatatga agttcatcat caaaaattgg tgttctttgc agaagatgtg 1860ggttcaaaca aaggtgcaat cattggactc atggtgggcg gtgttgtcat agcgacagtg 1920atcgtcatca ccttggtgat gctgaagaag aaacagtaca catccattca tcatggtgtg 1980gtggaggttg acgccgctgt caccccagag gagcgccacc tgtccaagat gcagcagaac 2040ggctacgaaa atccaaccta caagttcttt gagcagatgc agaactag 2088 12 695 PRTHomo sapiens 12 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp ThrAla Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu LeuAla Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His MetAsn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys ThrCys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val TyrPro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro ValThr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr HisPro His Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe ValSer Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His GlnGlu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr ValAla Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His AspTyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly ValGlu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val AspSer Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly GlyAla Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val GluVal Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 GluAla Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys AlaLys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser GlnVal Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn LeuPro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile Gln His Phe Gln Glu LysVal Glu Ser Leu Glu 355 360 365 Gln Glu Ala Ala Asn Glu Arg Gln Gln LeuVal Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp ArgArg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu Gln AlaVal Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys TyrVal Arg Ala Glu Gln Lys Asp Arg Gln His 420 425 430 Thr Leu Lys His PheGlu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala Gln Ile ArgSer Gln Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met AsnGln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 GluGlu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn 485 490 495Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr GluAsn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp ArgGly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys ThrGlu Glu Ile Ser 580 585 590 Glu Val Asn Leu Asp Ala Glu Phe Arg His AspSer Gly Tyr Glu Val 595 600 605 His His Gln Lys Leu Val Phe Phe Ala GluAsp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val GlyGly Val Val Ile Ala Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val MetLeu Lys Lys Lys Gln Tyr Thr Ser Ile 645 650 655 His His Gly Val Val GluVal Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys MetGln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu GlnMet Gln Asn 690 695 13 2088 PRT Homo sapiens 13 Ala Thr Gly Cys Thr GlyCys Cys Cys Gly Gly Thr Thr Thr Gly Gly 1 5 10 15 Cys Ala Cys Thr GlyCys Thr Cys Cys Thr Gly Cys Thr Gly Gly Cys 20 25 30 Cys Gly Cys Cys ThrGly Gly Ala Cys Gly Gly Cys Thr Cys Gly Gly 35 40 45 Gly Cys Gly Cys ThrGly Gly Ala Gly Gly Thr Ala Cys Cys Cys Ala 50 55 60 Cys Thr Gly Ala ThrGly Gly Thr Ala Ala Thr Gly Cys Thr Gly Gly 65 70 75 80 Cys Cys Thr GlyCys Thr Gly Gly Cys Thr Gly Ala Ala Cys Cys Cys 85 90 95 Cys Ala Gly AlaThr Thr Gly Cys Cys Ala Thr Gly Thr Thr Cys Thr 100 105 110 Gly Thr GlyGly Cys Ala Gly Ala Cys Thr Gly Ala Ala Cys Ala Thr 115 120 125 Gly CysAla Cys Ala Thr Gly Ala Ala Thr Gly Thr Cys Cys Ala Gly 130 135 140 AlaAla Thr Gly Gly Gly Ala Ala Gly Thr Gly Gly Gly Ala Thr Thr 145 150 155160 Cys Ala Gly Ala Thr Cys Cys Ala Thr Cys Ala Gly Gly Gly Ala Cys 165170 175 Cys Ala Ala Ala Ala Cys Cys Thr Gly Cys Ala Thr Thr Gly Ala Thr180 185 190 Ala Cys Cys Ala Ala Gly Gly Ala Ala Gly Gly Cys Ala Thr CysCys 195 200 205 Thr Gly Cys Ala Gly Thr Ala Thr Thr Gly Cys Cys Ala AlaGly Ala 210 215 220 Ala Gly Thr Cys Thr Ala Cys Cys Cys Thr Gly Ala AlaCys Thr Gly 225 230 235 240 Cys Ala Gly Ala Thr Cys Ala Cys Cys Ala AlaThr Gly Thr Gly Gly 245 250 255 Thr Ala Gly Ala Ala Gly Cys Cys Ala AlaCys Cys Ala Ala Cys Cys 260 265 270 Ala Gly Thr Gly Ala Cys Cys Ala ThrCys Cys Ala Gly Ala Ala Cys 275 280 285 Thr Gly Gly Thr Gly Cys Ala AlaGly Cys Gly Gly Gly Gly Cys Cys 290 295 300 Gly Cys Ala Ala Gly Cys AlaGly Thr Gly Cys Ala Ala Gly Ala Cys 305 310 315 320 Cys Cys Ala Thr CysCys Cys Cys Ala Cys Thr Thr Thr Gly Thr Gly 325 330 335 Ala Thr Thr CysCys Cys Thr Ala Cys Cys Gly Cys Thr Gly Cys Thr 340 345 350 Thr Ala GlyThr Thr Gly Gly Thr Gly Ala Gly Thr Thr Thr Gly Thr 355 360 365 Ala AlaGly Thr Gly Ala Thr Gly Cys Cys Cys Thr Thr Cys Thr Cys 370 375 380 GlyThr Thr Cys Cys Thr Gly Ala Cys Ala Ala Gly Thr Gly Cys Ala 385 390 395400 Ala Ala Thr Thr Cys Thr Thr Ala Cys Ala Cys Cys Ala Gly Gly Ala 405410 415 Gly Ala Gly Gly Ala Thr Gly Gly Ala Thr Gly Thr Thr Thr Gly Cys420 425 430 Gly Ala Ala Ala Cys Thr Cys Ala Thr Cys Thr Thr Cys Ala CysThr 435 440 445 Gly Gly Cys Ala Cys Ala Cys Cys Gly Thr Cys Gly Cys CysAla Ala 450 455 460 Ala Gly Ala Gly Ala Cys Ala Thr Gly Cys Ala Gly ThrGly Ala Gly 465 470 475 480 Ala Ala Gly Ala Gly Thr Ala Cys Cys Ala AlaCys Thr Thr Gly Cys 485 490 495 Ala Thr Gly Ala Cys Thr Ala Cys Gly GlyCys Ala Thr Gly Thr Thr 500 505 510 Gly Cys Thr Gly Cys Cys Cys Thr GlyCys Gly Gly Ala Ala Thr Thr 515 520 525 Gly Ala Cys Ala Ala Gly Thr ThrCys Cys Gly Ala Gly Gly Gly Gly 530 535 540 Thr Ala Gly Ala Gly Thr ThrThr Gly Thr Gly Thr Gly Thr Thr Gly 545 550 555 560 Cys Cys Cys Ala CysThr Gly Gly Cys Thr Gly Ala Ala Gly Ala Ala 565 570 575 Ala Gly Thr GlyAla Cys Ala Ala Thr Gly Thr Gly Gly Ala Thr Thr 580 585 590 Cys Thr GlyCys Thr Gly Ala Thr Gly Cys Gly Gly Ala Gly Gly Ala 595 600 605 Gly GlyAla Thr Gly Ala Cys Thr Cys Gly Gly Ala Thr Gly Thr Cys 610 615 620 ThrGly Gly Thr Gly Gly Gly Gly Cys Gly Gly Ala Gly Cys Ala Gly 625 630 635640 Ala Cys Ala Cys Ala Gly Ala Cys Thr Ala Thr Gly Cys Ala Gly Ala 645650 655 Thr Gly Gly Gly Ala Gly Thr Gly Ala Ala Gly Ala Cys Ala Ala Ala660 665 670 Gly Thr Ala Gly Thr Ala Gly Ala Ala Gly Thr Ala Gly Cys AlaGly 675 680 685 Ala Gly Gly Ala Gly Gly Ala Ala Gly Ala Ala Gly Thr GlyGly Cys 690 695 700 Thr Gly Ala Gly Gly Thr Gly Gly Ala Ala Gly Ala AlaGly Ala Ala 705 710 715 720 Gly Ala Ala Gly Cys Cys Gly Ala Thr Gly AlaThr Gly Ala Cys Gly 725 730 735 Ala Gly Gly Ala Cys Gly Ala Thr Gly AlaGly Gly Ala Thr Gly Gly 740 745 750 Thr Gly Ala Thr Gly Ala Gly Gly ThrAla Gly Ala Gly Gly Ala Ala 755 760 765 Gly Ala Gly Gly Cys Thr Gly AlaGly Gly Ala Ala Cys Cys Cys Thr 770 775 780 Ala Cys Gly Ala Ala Gly AlaAla Gly Cys Cys Ala Cys Ala Gly Ala 785 790 795 800 Gly Ala Gly Ala AlaCys Cys Ala Cys Cys Ala Gly Cys Ala Thr Thr 805 810 815 Gly Cys Cys AlaCys Cys Ala Cys Cys Ala Cys Cys Ala Cys Cys Ala 820 825 830 Cys Cys AlaCys Cys Ala Cys Ala Gly Ala Gly Thr Cys Thr Gly Thr 835 840 845 Gly GlyAla Ala Gly Ala Gly Gly Thr Gly Gly Thr Thr Cys Gly Ala 850 855 860 GlyThr Thr Cys Cys Thr Ala Cys Ala Ala Cys Ala Gly Cys Ala Gly 865 870 875880 Cys Cys Ala Gly Thr Ala Cys Cys Cys Cys Thr Gly Ala Thr Gly Cys 885890 895 Cys Gly Thr Thr Gly Ala Cys Ala Ala Gly Thr Ala Thr Cys Thr Cys900 905 910 Gly Ala Gly Ala Cys Ala Cys Cys Thr Gly Gly Gly Gly Ala ThrGly 915 920 925 Ala Gly Ala Ala Thr Gly Ala Ala Cys Ala Thr Gly Cys CysCys Ala 930 935 940 Thr Thr Thr Cys Cys Ala Gly Ala Ala Ala Gly Cys CysAla Ala Ala 945 950 955 960 Gly Ala Gly Ala Gly Gly Cys Thr Thr Gly AlaGly Gly Cys Cys Ala 965 970 975 Ala Gly Cys Ala Cys Cys Gly Ala Gly AlaGly Ala Gly Ala Ala Thr 980 985 990 Gly Thr Cys Cys Cys Ala Gly Gly ThrCys Ala Thr Gly Ala Gly Ala 995 1000 1005 Gly Ala Ala Thr Gly Gly GlyAla Ala Gly Ala Gly Gly Cys Ala 1010 1015 1020 Gly Ala Ala Cys Gly ThrCys Ala Ala Gly Cys Ala Ala Ala Gly 1025 1030 1035 Ala Ala Cys Thr ThrGly Cys Cys Thr Ala Ala Ala Gly Cys Thr 1040 1045 1050 Gly Ala Thr AlaAla Gly Ala Ala Gly Gly Cys Ala Gly Thr Thr 1055 1060 1065 Ala Thr CysCys Ala Gly Cys Ala Thr Thr Thr Cys Cys Ala Gly 1070 1075 1080 Gly AlaGly Ala Ala Ala Gly Thr Gly Gly Ala Ala Thr Cys Thr 1085 1090 1095 ThrThr Gly Gly Ala Ala Cys Ala Gly Gly Ala Ala Gly Cys Ala 1100 1105 1110Gly Cys Cys Ala Ala Cys Gly Ala Gly Ala Gly Ala Cys Ala Gly 1115 11201125 Cys Ala Gly Cys Thr Gly Gly Thr Gly Gly Ala Gly Ala Cys Ala 11301135 1140 Cys Ala Cys Ala Thr Gly Gly Cys Cys Ala Gly Ala Gly Thr Gly1145 1150 1155 Gly Ala Ala Gly Cys Cys Ala Thr Gly Cys Thr Cys Ala AlaThr 1160 1165 1170 Gly Ala Cys Cys Gly Cys Cys Gly Cys Cys Gly Cys CysThr Gly 1175 1180 1185 Gly Cys Cys Cys Thr Gly Gly Ala Gly Ala Ala CysThr Ala Cys 1190 1195 1200 Ala Thr Cys Ala Cys Cys Gly Cys Thr Cys ThrGly Cys Ala Gly 1205 1210 1215 Gly Cys Thr Gly Thr Thr Cys Cys Thr CysCys Thr Cys Gly Gly 1220 1225 1230 Cys Cys Thr Cys Gly Thr Cys Ala CysGly Thr Gly Thr Thr Cys 1235 1240 1245 Ala Ala Thr Ala Thr Gly Cys ThrAla Ala Ala Gly Ala Ala Gly 1250 1255 1260 Thr Ala Thr Gly Thr Cys CysGly Cys Gly Cys Ala Gly Ala Ala 1265 1270 1275 Cys Ala Gly Ala Ala GlyGly Ala Cys Ala Gly Ala Cys Ala Gly 1280 1285 1290 Cys Ala Cys Ala CysCys Cys Thr Ala Ala Ala Gly Cys Ala Thr 1295 1300 1305 Thr Thr Cys GlyAla Gly Cys Ala Thr Gly Thr Gly Cys Gly Cys 1310 1315 1320 Ala Thr GlyGly Thr Gly Gly Ala Thr Cys Cys Cys Ala Ala Gly 1325 1330 1335 Ala AlaAla Gly Cys Cys Gly Cys Thr Cys Ala Gly Ala Thr Cys 1340 1345 1350 CysGly Gly Thr Cys Cys Cys Ala Gly Gly Thr Thr Ala Thr Gly 1355 1360 1365Ala Cys Ala Cys Ala Cys Cys Thr Cys Cys Gly Thr Gly Thr Gly 1370 13751380 Ala Thr Thr Thr Ala Thr Gly Ala Gly Cys Gly Cys Ala Thr Gly 13851390 1395 Ala Ala Thr Cys Ala Gly Thr Cys Thr Cys Thr Cys Thr Cys Cys1400 1405 1410 Cys Thr Gly Cys Thr Cys Thr Ala Cys Ala Ala Cys Gly ThrGly 1415 1420 1425 Cys Cys Thr Gly Cys Ala Gly Thr Gly Gly Cys Cys GlyAla Gly 1430 1435 1440 Gly Ala Gly Ala Thr Thr Cys Ala Gly Gly Ala ThrGly Ala Ala 1445 1450 1455 Gly Thr Thr Gly Ala Thr Gly Ala Gly Cys ThrGly Cys Thr Thr 1460 1465 1470 Cys Ala Gly Ala Ala Ala Gly Ala Gly CysAla Ala Ala Ala Cys 1475 1480 1485 Thr Ala Thr Thr Cys Ala Gly Ala ThrGly Ala Cys Gly Thr Cys 1490 1495 1500 Thr Thr Gly Gly Cys Cys Ala AlaCys Ala Thr Gly Ala Thr Thr 1505 1510 1515 Ala Gly Thr Gly Ala Ala CysCys Ala Ala Gly Gly Ala Thr Cys 1520 1525 1530 Ala Gly Thr Thr Ala CysGly Gly Ala Ala Ala Cys Gly Ala Thr 1535 1540 1545 Gly Cys Thr Cys ThrCys Ala Thr Gly Cys Cys Ala Thr Cys Thr 1550 1555 1560 Thr Thr Gly AlaCys Cys Gly Ala Ala Ala Cys Gly Ala Ala Ala 1565 1570 1575 Ala Cys CysAla Cys Cys Gly Thr Gly Gly Ala Gly Cys Thr Cys 1580 1585 1590 Cys ThrThr Cys Cys Cys Gly Thr Gly Ala Ala Thr Gly Gly Ala 1595 1600 1605 GlyAla Gly Thr Thr Cys Ala Gly Cys Cys Thr Gly Gly Ala Cys 1610 1615 1620Gly Ala Thr Cys Thr Cys Cys Ala Gly Cys Cys Gly Thr Gly Gly 1625 16301635 Cys Ala Thr Thr Cys Thr Thr Thr Thr Gly Gly Gly Gly Cys Thr 16401645 1650 Gly Ala Cys Thr Cys Thr Gly Thr Gly Cys Cys Ala Gly Cys Cys1655 1660 1665 Ala Ala Cys Ala Cys Ala Gly Ala Ala Ala Ala Cys Gly AlaAla 1670 1675 1680 Gly Thr Thr Gly Ala Gly Cys Cys Thr Gly Thr Thr GlyAla Thr 1685 1690 1695 Gly Cys Cys Cys Gly Cys Cys Cys Thr Gly Cys ThrGly Cys Cys 1700 1705 1710 Gly Ala Cys Cys Gly Ala Gly Gly Ala Cys ThrGly Ala Cys Cys 1715 1720 1725 Ala Cys Thr Cys Gly Ala Cys Cys Ala GlyGly Thr Thr Cys Thr 1730 1735 1740 Gly Gly Gly Thr Thr Gly Ala Cys AlaAla Ala Thr Ala Thr Cys 1745 1750 1755 Ala Ala Gly Ala Cys Gly Gly AlaGly Gly Ala Gly Ala Thr Cys 1760 1765 1770 Thr Cys Thr Gly Ala Ala GlyThr Gly Ala Ala Gly Ala Thr Gly 1775 1780 1785 Gly Ala Thr Gly Cys AlaGly Ala Ala Thr Thr Cys Cys Gly Ala 1790 1795 1800 Cys Ala Thr Gly AlaCys Thr Cys Ala Gly Gly Ala Thr Ala Thr 1805 1810 1815 Gly Ala Ala GlyThr Thr Cys Ala Thr Cys Ala Thr Cys Ala Ala 1820 1825 1830 Ala Ala AlaThr Thr Gly Gly Thr Gly Thr Thr Cys Thr Thr Thr 1835 1840 1845 Gly CysAla Gly Ala Ala Gly Ala Thr Gly Thr Gly Gly Gly Thr 1850 1855 1860 ThrCys Ala Ala Ala Cys Ala Ala Ala Gly Gly Thr Gly Cys Ala 1865 1870 1875Ala Thr Cys Ala Thr Thr Gly Gly Ala Cys Thr Cys Ala Thr Gly 1880 18851890 Gly Thr Gly Gly Gly Cys Gly Gly Thr Gly Thr Thr Gly Thr Cys 18951900 1905 Ala Thr Ala Gly Cys Gly Ala Cys Ala Gly Thr Gly Ala Thr Cys1910 1915 1920 Thr Thr Cys Ala Thr Cys Ala Cys Cys Thr Thr Gly Gly ThrGly 1925 1930 1935 Ala Thr Gly Cys Thr Gly Ala Ala Gly Ala Ala Gly AlaAla Ala 1940 1945 1950 Cys Ala Gly Thr Ala Cys Ala Cys Ala Thr Cys CysAla Thr Thr 1955 1960 1965 Cys Ala Thr Cys Ala Thr Gly Gly Thr Gly ThrGly Gly Thr Gly 1970 1975 1980 Gly Ala Gly Gly Thr Thr Gly Ala Cys GlyCys Cys Gly Cys Thr 1985 1990 1995 Gly Thr Cys Ala Cys Cys Cys Cys AlaGly Ala Gly Gly Ala Gly 2000 2005 2010 Cys Gly Cys Cys Ala Cys Cys ThrGly Thr Cys Cys Ala Ala Gly 2015 2020 2025 Ala Thr Gly Cys Ala Gly CysAla Gly Ala Ala Cys Gly Gly Cys 2030 2035 2040 Thr Ala Cys Gly Ala AlaAla Ala Thr Cys Cys Ala Ala Cys Cys 2045 2050 2055 Thr Ala Cys Ala AlaGly Thr Thr Cys Thr Thr Thr Gly Ala Gly 2060 2065 2070 Cys Ala Gly AlaThr Gly Cys Ala Gly Ala Ala Cys Thr Ala Gly 2075 2080 2085 14 695 PRTHomo sapiens 14 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp ThrAla Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu LeuAla Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His MetAsn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys ThrCys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val TyrPro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro ValThr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr HisPro His Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe ValSer Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His GlnGlu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr ValAla Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His AspTyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly ValGlu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val AspSer Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly GlyAla Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val GluVal Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 GluAla Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275280 285 Val Pro Thr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu290 295 300 Glu Thr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys AlaLys 305 310 315 320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser GlnVal Met Arg 325 330 335 Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn LeuPro Lys Ala Asp 340 345 350 Lys Lys Ala Val Ile Gln His Phe Gln Glu LysVal Glu Ser Leu Glu 355 360 365 Gln Glu Ala Ala Asn Glu Arg Gln Gln LeuVal Glu Thr His Met Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp ArgArg Arg Leu Ala Leu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu Gln AlaVal Pro Pro Arg Pro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys TyrVal Arg Ala Glu Gln Lys Asp Arg Gln His 420 425 430 Thr Leu Lys His PheGlu His Val Arg Met Val Asp Pro Lys Lys Ala 435 440 445 Ala Gln Ile ArgSer Gln Val Met Thr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met AsnGln Ser Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 GluGlu Ile Gln Asp Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn 485 490 495Tyr Ser Asp Asp Val Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505510 Tyr Gly Asn Asp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515520 525 Val Glu Leu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln530 535 540 Pro Trp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr GluAsn 545 550 555 560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp ArgGly Leu Thr 565 570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys ThrGlu Glu Ile Ser 580 585 590 Glu Val Lys Met Asp Ala Glu Phe Arg His AspSer Gly Tyr Glu Val 595 600 605 His His Gln Lys Leu Val Phe Phe Ala GluAsp Val Gly Ser Asn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val GlyGly Val Val Ile Ala Thr Val 625 630 635 640 Ile Phe Ile Thr Leu Val MetLeu Lys Lys Lys Gln Tyr Thr Ser Ile 645 650 655 His His Gly Val Val GluVal Asp Ala Ala Val Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys MetGln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu GlnMet Gln Asn 690 695 15 2094 DNA Homo sapiens 15 atgctgcccg gtttggcactgctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctggcctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgtccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaaggaaggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtagaagccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagacccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgcccttctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactcatcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatgactacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttgcccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcggatgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtagaagtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgaggacgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacagagagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggttcgagttcct acaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacctggggatgag aatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagcaccgagagaga atgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaacttgcctaaa gctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatctttggaacaggaa gcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtggaagccatgctc aatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggctgttcctcct cggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaacagaaggacaga cagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaaagccgctcag atccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaatcagtctctc tccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagttgatgagctg cttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattagtgaaccaagg atcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaaccaccgtggag ctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggcattcttttggg gctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgcccgccctgct gccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaagacggaggag atctctgaag tgaagatgga tgcagaattc 1800 cgacatgact caggatatgaagttcatcat caaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaatcattggactc atggtgggcg gtgttgtcat agcgacagtg 1920 atcgtcatca ccttggtgatgctgaagaag aaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgtcaccccagag gagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaacctacaagttcttt gagcagatgc agaacaagaa gtag 2094 16 697 PRT Homo sapines 16Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 1015 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 2530 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 4045 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 5560 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 7075 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 8590 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala LeuLeu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met AspVal Cys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu ThrCys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met LeuLeu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val CysCys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp AlaGlu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp Thr AspTyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu GluGlu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp AspGlu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu GluPro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr ThrThr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val ProThr Thr Ala Ala Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 GluThr Pro Gly Asp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys 305 310 315320 Glu Arg Leu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg 325330 335 Glu Trp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp340 345 350 Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser LeuGlu 355 360 365 Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr HisMet Ala 370 375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu AlaLeu Glu Asn 385 390 395 400 Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro ArgPro Arg His Val Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala GluGln Lys Asp Arg Gln His 420 425 430 Thr Leu Lys His Phe Glu His Val ArgMet Val Asp Pro Lys Lys Ala 435 440 445 Ala Gln Ile Arg Ser Gln Val MetThr His Leu Arg Val Ile Tyr Glu 450 455 460 Arg Met Asn Gln Ser Leu SerLeu Leu Tyr Asn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile Gln AspGlu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn 485 490 495 Tyr Ser Asp AspVal Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly AsnAsp Ala Leu Met Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val GluLeu Leu Pro Val Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln 530 535 540 ProTrp His Ser Phe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555560 Glu Val Glu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565570 575 Thr Arg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser580 585 590 Glu Val Lys Met Asp Ala Glu Phe Arg His Asp Ser Gly Tyr GluVal 595 600 605 His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly SerAsn Lys 610 615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val IleAla Thr Val 625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys Lys LysGln Tyr Thr Ser Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala AlaVal Thr Pro Glu Glu Arg 660 665 670 His Leu Ser Lys Met Gln Gln Asn GlyTyr Glu Asn Pro Thr Tyr Lys 675 680 685 Phe Phe Glu Gln Met Gln Asn LysLys 690 695 17 2094 DNA Homo sapiens 17 atgctgcccg gtttggcact gctcctgctggccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggctgaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatgggaagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctgcagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaaccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccactttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcctgacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactggcacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatgttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggctgaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtggggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagaggaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggatggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccaccagcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcctacaacagcag ccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgagaatgaacatg cccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagagaatgtcccagg tcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaagctgataaga aggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaagcagccaacg agagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctcaatgaccgcc gccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcctcggcctcgtc acgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacagacagcacaccc taaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcagatccggtccc aggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctctccctgctct acaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctgcttcagaaag agcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaaggatcagttacg gaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggagctccttcccg tgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggggctgactctg tgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgctgccgaccgag gactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggagatctctgaag tgaatctgga tgcagaattc 1800 cgacatgact caggatatga agttcatcatcaaaaattgg tgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactcatggtgggcg gtgttgtcat agcgacagtg 1920 atcgtcatca ccttggtgat gctgaagaagaaacagtaca catccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagaggagcgccacc tgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttctttgagcagatgc agaacaagaa gtag 2094 18 697 PRT Homo sapiens 18 Met Leu ProGly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala LeuGlu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln IleAla Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45 Asn GlyLys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr LysGlu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 GlnIle Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 TrpCys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro CysGly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro LeuAla Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu AspAsp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala AspGly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu ValAla Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp AspGlu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr GluGlu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr ThrThr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr AlaAla Ser Thr Pro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro GlyAsp Glu Asn Glu His Ala His Phe Gln Lys Ala Lys 305 310 315 320 Glu ArgLeu Glu Ala Lys His Arg Glu Arg Met Ser Gln Val Met Arg 325 330 335 GluTrp Glu Glu Ala Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350Lys Lys Ala Val Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu 355 360365 Gln Glu Ala Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala 370375 380 Arg Val Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn385 390 395 400 Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg HisVal Phe 405 410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys AspArg Gln His 420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val AspPro Lys Lys Ala 435 440 445 Ala Gln Ile Arg Ser Gln Val Met Thr His LeuArg Val Ile Tyr Glu 450 455 460 Arg Met Asn Gln Ser Leu Ser Leu Leu TyrAsn Val Pro Ala Val Ala 465 470 475 480 Glu Glu Ile Gln Asp Glu Val AspGlu Leu Leu Gln Lys Glu Gln Asn 485 490 495 Tyr Ser Asp Asp Val Leu AlaAsn Met Ile Ser Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala LeuMet Pro Ser Leu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu ProVal Asn Gly Glu Phe Ser Leu Asp Asp Leu Gln 530 535 540 Pro Trp His SerPhe Gly Ala Asp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu ValGlu Pro Val Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 ThrArg Pro Gly Ser Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590Glu Val Asn Leu Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600605 His His Gln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610615 620 Gly Ala Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val625 630 635 640 Ile Val Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr ThrSer Ile 645 650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr ProGlu Glu Arg 660 665 670 His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu AsnPro Thr Tyr Lys 675 680 685 Phe Phe Glu Gln Met Gln Asn Lys Lys 690 69519 2094 DNA Homo sapiens 19 atgctgcccg gtttggcact gctcctgctg gccgcctggacggctcgggc gctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccagattgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggattcagatccatc agggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgccaagaagtcta ccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgaccatccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattccctaccgctg cttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgcaaattcttaca ccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcgccaaagagac atgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccctgcggaattga caagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtgacaatgtgga ttctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcagacacagacta tgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaagtggctgaggt ggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgaggtagaggaaga ggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgccaccaccaccac caccaccaca 840 gagtctgtgg aagaggtggt tcgagttcct acaacagcagccagtacccc tgatgccgtt 900 gacaagtatc tcgagacacc tggggatgag aatgaacatgcccatttcca gaaagccaaa 960 gagaggcttg aggccaagca ccgagagaga atgtcccaggtcatgagaga atgggaagag 1020 gcagaacgtc aagcaaagaa cttgcctaaa gctgataagaaggcagttat ccagcatttc 1080 caggagaaag tggaatcttt ggaacaggaa gcagccaacgagagacagca gctggtggag 1140 acacacatgg ccagagtgga agccatgctc aatgaccgccgccgcctggc cctggagaac 1200 tacatcaccg ctctgcaggc tgttcctcct cggcctcgtcacgtgttcaa tatgctaaag 1260 aagtatgtcc gcgcagaaca gaaggacaga cagcacaccctaaagcattt cgagcatgtg 1320 cgcatggtgg atcccaagaa agccgctcag atccggtcccaggttatgac acacctccgt 1380 gtgatttatg agcgcatgaa tcagtctctc tccctgctctacaacgtgcc tgcagtggcc 1440 gaggagattc aggatgaagt tgatgagctg cttcagaaagagcaaaacta ttcagatgac 1500 gtcttggcca acatgattag tgaaccaagg atcagttacggaaacgatgc tctcatgcca 1560 tctttgaccg aaacgaaaac caccgtggag ctccttcccgtgaatggaga gttcagcctg 1620 gacgatctcc agccgtggca ttcttttggg gctgactctgtgccagccaa cacagaaaac 1680 gaagttgagc ctgttgatgc ccgccctgct gccgaccgaggactgaccac tcgaccaggt 1740 tctgggttga caaatatcaa gacggaggag atctctgaagtgaagatgga tgcagaattc 1800 cgacatgact caggatatga agttcatcat caaaaattggtgttctttgc agaagatgtg 1860 ggttcaaaca aaggtgcaat cattggactc atggtgggcggtgttgtcat agcgacagtg 1920 atcttcatca ccttggtgat gctgaagaag aaacagtacacatccattca tcatggtgtg 1980 gtggaggttg acgccgctgt caccccagag gagcgccacctgtccaagat gcagcagaac 2040 ggctacgaaa atccaaccta caagttcttt gagcagatgcagaacaagaa gtag 2094 20 697 PRT Homo sapiens 20 Met Leu Pro Gly Leu AlaLeu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val ProThr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met PheCys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp AspSer Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly IleLeu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr AsnVal Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys ArgGly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110 Ile Pro TyrArg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 Val ProAsp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 GluThr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser AspVal 195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser GluAsp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu ValGlu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp GlyAsp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala ThrGlu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr GluSer Val Glu Glu Val Val Arg 275 280 285 Val Pro Thr Thr Ala Ala Ser ThrPro Asp Ala Val Asp Lys Tyr Leu 290 295 300 Glu Thr Pro Gly Asp Glu AsnGlu His Ala His Phe Gln Lys Ala Lys 305 310 315 320 Glu Arg Leu Glu AlaLys His Arg Glu Arg Met Ser Gln Val Met Arg 325 330 335 Glu Trp Glu GluAla Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp 340 345 350 Lys Lys AlaVal Ile Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu 355 360 365 Gln GluAla Ala Asn Glu Arg Gln Gln Leu Val Glu Thr His Met Ala 370 375 380 ArgVal Glu Ala Met Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn 385 390 395400 Tyr Ile Thr Ala Leu Gln Ala Val Pro Pro Arg Pro Arg His Val Phe 405410 415 Asn Met Leu Lys Lys Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His420 425 430 Thr Leu Lys His Phe Glu His Val Arg Met Val Asp Pro Lys LysAla 435 440 445 Ala Gln Ile Arg Ser Gln Val Met Thr His Leu Arg Val IleTyr Glu 450 455 460 Arg Met Asn Gln Ser Leu Ser Leu Leu Tyr Asn Val ProAla Val Ala 465 470 475 480 Glu Glu Ile Gln Asp Glu Val Asp Glu Leu LeuGln Lys Glu Gln Asn 485 490 495 Tyr Ser Asp Asp Val Leu Ala Asn Met IleSer Glu Pro Arg Ile Ser 500 505 510 Tyr Gly Asn Asp Ala Leu Met Pro SerLeu Thr Glu Thr Lys Thr Thr 515 520 525 Val Glu Leu Leu Pro Val Asn GlyGlu Phe Ser Leu Asp Asp Leu Gln 530 535 540 Pro Trp His Ser Phe Gly AlaAsp Ser Val Pro Ala Asn Thr Glu Asn 545 550 555 560 Glu Val Glu Pro ValAsp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr 565 570 575 Thr Arg Pro GlySer Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser 580 585 590 Glu Val LysMet Asp Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val 595 600 605 His HisGln Lys Leu Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys 610 615 620 GlyAla Ile Ile Gly Leu Met Val Gly Gly Val Val Ile Ala Thr Val 625 630 635640 Ile Phe Ile Thr Leu Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile 645650 655 His His Gly Val Val Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg660 665 670 His Leu Ser Lys Met Gln Gln Asn Gly Tyr Glu Asn Pro Thr TyrLys 675 680 685 Phe Phe Glu Gln Met Gln Asn Lys Lys 690 695 21 1341 DNAHomo sapiens 21 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccacccagcacggcatccgg 60 ctgcccctgc gcagcggcct ggggggcgcc cccctggggc tgcggctgccccgggagacc 120 gacgaagagc ccgaggagcc cggccggagg ggcagctttg tggagatggtggacaacctg 180 aggggcaagt cggggcaggg ctactacgtg gagatgaccg tgggcagccccccgcagacg 240 ctcaacatcc tggtggatac aggcagcagt aactttgcag tgggtgctgccccccacccc 300 ttcctgcatc gctactacca gaggcagctg tccagcacat accgggacctccggaagggt 360 gtgtatgtgc cctacaccca gggcaagtgg gaaggggagc tgggcaccgacctggtaagc 420 atcccccatg gccccaacgt cactgtgcgt gccaacattg ctgccatcactgaatcagac 480 aagttcttca tcaacggctc caactgggaa ggcatcctgg ggctggcctatgctgagatt 540 gccaggcctg acgactccct ggagcctttc tttgactctc tggtaaagcagacccacgtt 600 cccaacctct tctccctgca cctttgtggt gctggcttcc ccctcaaccagtctgaagtg 660 ctggcctctg tcggagggag catgatcatt ggaggtatcg accactcgctgtacacaggc 720 agtctctggt atacacccat ccggcgggag tggtattatg aggtcatcattgtgcgggtg 780 gagatcaatg gacaggatct gaaaatggac tgcaaggagt acaactatgacaagagcatt 840 gtggacagtg gcaccaccaa ccttcgtttg cccaagaaag tgtttgaagctgcagtcaaa 900 tccatcaagg cagcctcctc cacggagaag ttccctgatg gtttctggctaggagagcag 960 ctggtgtgct ggcaagcagg caccacccct tggaacattt tcccagtcatctcactctac 1020 ctaatgggtg aggttaccaa ccagtccttc cgcatcacca tccttccgcagcaatacctg 1080 cggccagtgg aagatgtggc cacgtcccaa gacgactgtt acaagtttgccatctcacag 1140 tcatccacgg gcactgttat gggagctgtt atcatggagg gcttctacgttgtctttgat 1200 cgggcccgaa aacgaattgg ctttgctgtc agcgcttgcc atgtgcacgatgagttcagg 1260 acggcagcgg tggaaggccc ttttgtcacc ttggacatgg aagactgtggctacaacatt 1320 ccacagacag atgagtcatg a 1341 22 446 PRT Homo sapiens 22Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Thr Gln 1 5 1015 His Gly Ile Arg Leu Pro Leu Arg Ser Gly Leu Gly Gly Ala Pro Leu 20 2530 Gly Leu Arg Leu Pro Arg Glu Thr Asp Glu Glu Pro Glu Glu Pro Gly 35 4045 Arg Arg Gly Ser Phe Val Glu Met Val Asp Asn Leu Arg Gly Lys Ser 50 5560 Gly Gln Gly Tyr Tyr Val Glu Met Thr Val Gly Ser Pro Pro Gln Thr 65 7075 80 Leu Asn Ile Leu Val Asp Thr Gly Ser Ser Asn Phe Ala Val Gly Ala 8590 95 Ala Pro His Pro Phe Leu His Arg Tyr Tyr Gln Arg Gln Leu Ser Ser100 105 110 Thr Tyr Arg Asp Leu Arg Lys Gly Val Tyr Val Pro Tyr Thr GlnGly 115 120 125 Lys Trp Glu Gly Glu Leu Gly Thr Asp Leu Val Ser Ile ProHis Gly 130 135 140 Pro Asn Val Thr Val Arg Ala Asn Ile Ala Ala Ile ThrGlu Ser Asp 145 150 155 160 Lys Phe Phe Ile Asn Gly Ser Asn Trp Glu GlyIle Leu Gly Leu Ala 165 170 175 Tyr Ala Glu Ile Ala Arg Pro Asp Asp SerLeu Glu Pro Phe Phe Asp 180 185 190 Ser Leu Val Lys Gln Thr His Val ProAsn Leu Phe Ser Leu His Leu 195 200 205 Cys Gly Ala Gly Phe Pro Leu AsnGln Ser Glu Val Leu Ala Ser Val 210 215 220 Gly Gly Ser Met Ile Ile GlyGly Ile Asp His Ser Leu Tyr Thr Gly 225 230 235 240 Ser Leu Trp Tyr ThrPro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile 245 250 255 Ile Val Arg ValGlu Ile Asn Gly Gln Asp Leu Lys Met Asp Cys Lys 260 265 270 Glu Tyr AsnTyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu 275 280 285 Arg LeuPro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala 290 295 300 AlaSer Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu Gln 305 310 315320 Leu Val Cys Trp Gln Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val 325330 335 Ile Ser Leu Tyr Leu Met Gly Glu Val Thr Asn Gln Ser Phe Arg Ile340 345 350 Thr Ile Leu Pro Gln Gln Tyr Leu Arg Pro Val Glu Asp Val AlaThr 355 360 365 Ser Gln Asp Asp Cys Tyr Lys Phe Ala Ile Ser Gln Ser SerThr Gly 370 375 380 Thr Val Met Gly Ala Val Ile Met Glu Gly Phe Tyr ValVal Phe Asp 385 390 395 400 Arg Ala Arg Lys Arg Ile Gly Phe Ala Val SerAla Cys His Val His 405 410 415 Asp Glu Phe Arg Thr Ala Ala Val Glu GlyPro Phe Val Thr Leu Asp 420 425 430 Met Glu Asp Cys Gly Tyr Asn Ile ProGln Thr Asp Glu Ser 435 440 445 23 1380 DNA Homo sapiens 23 atggctagcatgactggtgg acagcaaatg ggtcgcggat cgatgactat ctctgactct 60 ccgcgtgaacaggacggatc cacccagcac ggcatccggc tgcccctgcg cagcggcctg 120 gggggcgcccccctggggct gcggctgccc cgggagaccg acgaagagcc cgaggagccc 180 ggccggaggggcagctttgt ggagatggtg gacaacctga ggggcaagtc ggggcagggc 240 tactacgtggagatgaccgt gggcagcccc ccgcagacgc tcaacatcct ggtggataca 300 ggcagcagtaactttgcagt gggtgctgcc ccccacccct tcctgcatcg ctactaccag 360 aggcagctgtccagcacata ccgggacctc cggaagggtg tgtatgtgcc ctacacccag 420 ggcaagtgggaaggggagct gggcaccgac ctggtaagca tcccccatgg ccccaacgtc 480 actgtgcgtgccaacattgc tgccatcact gaatcagaca agttcttcat caacggctcc 540 aactgggaaggcatcctggg gctggcctat gctgagattg ccaggcctga cgactccctg 600 gagcctttctttgactctct ggtaaagcag acccacgttc ccaacctctt ctccctgcac 660 ctttgtggtgctggcttccc cctcaaccag tctgaagtgc tggcctctgt cggagggagc 720 atgatcattggaggtatcga ccactcgctg tacacaggca gtctctggta tacacccatc 780 cggcgggagtggtattatga ggtcatcatt gtgcgggtgg agatcaatgg acaggatctg 840 aaaatggactgcaaggagta caactatgac aagagcattg tggacagtgg caccaccaac 900 cttcgtttgcccaagaaagt gtttgaagct gcagtcaaat ccatcaaggc agcctcctcc 960 acggagaagttccctgatgg tttctggcta ggagagcagc tggtgtgctg gcaagcaggc 1020 accaccccttggaacatttt cccagtcatc tcactctacc taatgggtga ggttaccaac 1080 cagtccttccgcatcaccat ccttccgcag caatacctgc ggccagtgga agatgtggcc 1140 acgtcccaagacgactgtta caagtttgcc atctcacagt catccacggg cactgttatg 1200 ggagctgttatcatggaggg cttctacgtt gtctttgatc gggcccgaaa acgaattggc 1260 tttgctgtcagcgcttgcca tgtgcacgat gagttcagga cggcagcggt ggaaggccct 1320 tttgtcaccttggacatgga agactgtggc tacaacattc cacagacaga tgagtcatga 1380 24 459 PRTHomo sapiens 24 Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly SerMet Thr 1 5 10 15 Ile Ser Asp Ser Pro Arg Glu Gln Asp Gly Ser Thr GlnHis Gly Ile 20 25 30 Arg Leu Pro Leu Arg Ser Gly Leu Gly Gly Ala Pro LeuGly Leu Arg 35 40 45 Leu Pro Arg Glu Thr Asp Glu Glu Pro Glu Glu Pro GlyArg Arg Gly 50 55 60 Ser Phe Val Glu Met Val Asp Asn Leu Arg Gly Lys SerGly Gln Gly 65 70 75 80 Tyr Tyr Val Glu Met Thr Val Gly Ser Pro Pro GlnThr Leu Asn Ile 85 90 95 Leu Val Asp Thr Gly Ser Ser Asn Phe Ala Val GlyAla Ala Pro His 100 105 110 Pro Phe Leu His Arg Tyr Tyr Gln Arg Gln LeuSer Ser Thr Tyr Arg 115 120 125 Asp Leu Arg Lys Gly Val Tyr Val Pro TyrThr Gln Gly Lys Trp Glu 130 135 140 Gly Glu Leu Gly Thr Asp Leu Val SerIle Pro His Gly Pro Asn Val 145 150 155 160 Thr Val Arg Ala Asn Ile AlaAla Ile Thr Glu Ser Asp Lys Phe Phe 165 170 175 Ile Asn Gly Ser Asn TrpGlu Gly Ile Leu Gly Leu Ala Tyr Ala Glu 180 185 190 Ile Ala Arg Pro AspAsp Ser Leu Glu Pro Phe Phe Asp Ser Leu Val 195 200 205 Lys Gln Thr HisVal Pro Asn Leu Phe Ser Leu His Leu Cys Gly Ala 210 215 220 Gly Phe ProLeu Asn Gln Ser Glu Val Leu Ala Ser Val Gly Gly Ser 225 230 235 240 MetIle Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu Trp 245 250 255Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg 260 265270 Val Glu Ile Asn Gly Gln Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn 275280 285 Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro290 295 300 Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala SerSer 305 310 315 320 Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu Gly Glu GlnLeu Val Cys 325 330 335 Trp Gln Ala Gly Thr Thr Pro Trp Asn Ile Phe ProVal Ile Ser Leu 340 345 350 Tyr Leu Met Gly Glu Val Thr Asn Gln Ser PheArg Ile Thr Ile Leu 355 360 365 Pro Gln Gln Tyr Leu Arg Pro Val Glu AspVal Ala Thr Ser Gln Asp 370 375 380 Asp Cys Tyr Lys Phe Ala Ile Ser GlnSer Ser Thr Gly Thr Val Met 385 390 395 400 Gly Ala Val Ile Met Glu GlyPhe Tyr Val Val Phe Asp Arg Ala Arg 405 410 415 Lys Arg Ile Gly Phe AlaVal Ser Ala Cys His Val His Asp Glu Phe 420 425 430 Arg Thr Ala Ala ValGlu Gly Pro Phe Val Thr Leu Asp Met Glu Asp 435 440 445 Cys Gly Tyr AsnIle Pro Gln Thr Asp Glu Ser 450 455 25 1302 DNA Homo sapiens 25atgactcagc atggtattcg tctgccactg cgtagcggtc tgggtggtgc tccactgggt 60ctgcgtctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 120gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 180gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 240gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 300taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 360ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 420gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 480gggctggcct atgctgagat tgccaggcct gacgactccc tggagccttt ctttgactct 540ctggtaaagc agacccacgt tcccaacctc ttctccctgc acctttgtgg tgctggcttc 600cccctcaacc agtctgaagt gctggcctct gtcggaggga gcatgatcat tggaggtatc 660gaccactcgc tgtacacagg cagtctctgg tatacaccca tccggcggga gtggtattat 720gaggtcatca ttgtgcgggt ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag 780tacaactatg acaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 840gtgtttgaag ctgcagtcaa atccatcaag gcagcctcct ccacggagaa gttccctgat 900ggtttctggc taggagagca gctggtgtgc tggcaagcag gcaccacccc ttggaacatt 960ttcccagtca tctcactcta cctaatgggt gaggttacca accagtcctt ccgcatcacc 1020atccttccgc agcaatacct gcggccagtg gaagatgtgg ccacgtccca agacgactgt 1080tacaagtttg ccatctcaca gtcatccacg ggcactgtta tgggagctgt tatcatggag 1140ggcttctacg ttgtctttga tcgggcccga aaacgaattg gctttgctgt cagcgcttgc 1200catgtgcacg atgagttcag gacggcagcg gtggaaggcc cttttgtcac cttggacatg 1260gaagactgtg gctacaacat tccacagaca gatgagtcat ga 1302 26 433 PRT Homosapiens 26 Met Thr Gln His Gly Ile Arg Leu Pro Leu Arg Ser Gly Leu GlyGly 1 5 10 15 Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp Glu GluPro Glu 20 25 30 Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val Asp AsnLeu Arg 35 40 45 Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr Val GlySer Pro 50 55 60 Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser Ser AsnPhe Ala 65 70 75 80 Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr TyrGln Arg Gln 85 90 95 Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val TyrVal Pro Tyr 100 105 110 Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr AspLeu Val Ser Ile 115 120 125 Pro His Gly Pro Asn Val Thr Val Arg Ala AsnIle Ala Ala Ile Thr 130 135 140 Glu Ser Asp Lys Phe Phe Ile Asn Gly SerAsn Trp Glu Gly Ile Leu 145 150 155 160 Gly Leu Ala Tyr Ala Glu Ile AlaArg Pro Asp Asp Ser Leu Glu Pro 165 170 175 Phe Phe Asp Ser Leu Val LysGln Thr His Val Pro Asn Leu Phe Ser 180 185 190 Leu His Leu Cys Gly AlaGly Phe Pro Leu Asn Gln Ser Glu Val Leu 195 200 205 Ala Ser Val Gly GlySer Met Ile Ile Gly Gly Ile Asp His Ser Leu 210 215 220 Tyr Thr Gly SerLeu Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr 225 230 235 240 Glu ValIle Ile Val Arg Val Glu Ile Asn Gly Gln Asp Leu Lys Met 245 250 255 AspCys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr 260 265 270Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser 275 280285 Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu 290295 300 Gly Glu Gln Leu Val Cys Trp Gln Ala Gly Thr Thr Pro Trp Asn Ile305 310 315 320 Phe Pro Val Ile Ser Leu Tyr Leu Met Gly Glu Val Thr AsnGln Ser 325 330 335 Phe Arg Ile Thr Ile Leu Pro Gln Gln Tyr Leu Arg ProVal Glu Asp 340 345 350 Val Ala Thr Ser Gln Asp Asp Cys Tyr Lys Phe AlaIle Ser Gln Ser 355 360 365 Ser Thr Gly Thr Val Met Gly Ala Val Ile MetGlu Gly Phe Tyr Val 370 375 380 Val Phe Asp Arg Ala Arg Lys Arg Ile GlyPhe Ala Val Ser Ala Cys 385 390 395 400 His Val His Asp Glu Phe Arg ThrAla Ala Val Glu Gly Pro Phe Val 405 410 415 Thr Leu Asp Met Glu Asp CysGly Tyr Asn Ile Pro Gln Thr Asp Glu 420 425 430 Ser 27 1278 DNA Homosapiens 27 atggctagca tgactggtgg acagcaaatg ggtcgcggat cgatgactatctctgactct 60 ccgctggact ctggtatcga aaccgacgga tcctttgtgg agatggtggacaacctgagg 120 ggcaagtcgg ggcagggcta ctacgtggag atgaccgtgg gcagccccccgcagacgctc 180 aacatcctgg tggatacagg cagcagtaac tttgcagtgg gtgctgccccccaccccttc 240 ctgcatcgct actaccagag gcagctgtcc agcacatacc gggacctccggaagggtgtg 300 tatgtgccct acacccaggg caagtgggaa ggggagctgg gcaccgacctggtaagcatc 360 ccccatggcc ccaacgtcac tgtgcgtgcc aacattgctg ccatcactgaatcagacaag 420 ttcttcatca acggctccaa ctgggaaggc atcctggggc tggcctatgctgagattgcc 480 aggcctgacg actccctgga gcctttcttt gactctctgg taaagcagacccacgttccc 540 aacctcttct ccctgcacct ttgtggtgct ggcttccccc tcaaccagtctgaagtgctg 600 gcctctgtcg gagggagcat gatcattgga ggtatcgacc actcgctgtacacaggcagt 660 ctctggtata cacccatccg gcgggagtgg tattatgagg tcatcattgtgcgggtggag 720 atcaatggac aggatctgaa aatggactgc aaggagtaca actatgacaagagcattgtg 780 gacagtggca ccaccaacct tcgtttgccc aagaaagtgt ttgaagctgcagtcaaatcc 840 atcaaggcag cctcctccac ggagaagttc cctgatggtt tctggctaggagagcagctg 900 gtgtgctggc aagcaggcac caccccttgg aacattttcc cagtcatctcactctaccta 960 atgggtgagg ttaccaacca gtccttccgc atcaccatcc ttccgcagcaatacctgcgg 1020 ccagtggaag atgtggccac gtcccaagac gactgttaca agtttgccatctcacagtca 1080 tccacgggca ctgttatggg agctgttatc atggagggct tctacgttgtctttgatcgg 1140 gcccgaaaac gaattggctt tgctgtcagc gcttgccatg tgcacgatgagttcaggacg 1200 gcagcggtgg aaggcccttt tgtcaccttg gacatggaag actgtggctacaacattcca 1260 cagacagatg agtcatga 1278 28 425 PRT Homo sapiens 28 MetAla Ser Met Thr Gly Gly Gln Gln Met Gly Arg Gly Ser Met Thr 1 5 10 15Ile Ser Asp Ser Pro Leu Asp Ser Gly Ile Glu Thr Asp Gly Ser Phe 20 25 30Val Glu Met Val Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr 35 40 45Val Glu Met Thr Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val 50 55 60Asp Thr Gly Ser Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe 65 70 7580 Leu His Arg Tyr Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu 85 9095 Arg Lys Gly Val Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu 100105 110 Leu Gly Thr Asp Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val115 120 125 Arg Ala Asn Ile Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe IleAsn 130 135 140 Gly Ser Asn Trp Glu Gly Ile Leu Gly Leu Ala Tyr Ala GluIle Ala 145 150 155 160 Arg Pro Asp Asp Ser Leu Glu Pro Phe Phe Asp SerLeu Val Lys Gln 165 170 175 Thr His Val Pro Asn Leu Phe Ser Leu His LeuCys Gly Ala Gly Phe 180 185 190 Pro Leu Asn Gln Ser Glu Val Leu Ala SerVal Gly Gly Ser Met Ile 195 200 205 Ile Gly Gly Ile Asp His Ser Leu TyrThr Gly Ser Leu Trp Tyr Thr 210 215 220 Pro Ile Arg Arg Glu Trp Tyr TyrGlu Val Ile Ile Val Arg Val Glu 225 230 235 240 Ile Asn Gly Gln Asp LeuLys Met Asp Cys Lys Glu Tyr Asn Tyr Asp 245 250 255 Lys Ser Ile Val AspSer Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys 260 265 270 Val Phe Glu AlaAla Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu 275 280 285 Lys Phe ProAsp Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln 290 295 300 Ala GlyThr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr Leu 305 310 315 320Met Gly Glu Val Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu Pro Gln 325 330335 Gln Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser Gln Asp Asp Cys 340345 350 Tyr Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr Val Met Gly Ala355 360 365 Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala Arg LysArg 370 375 380 Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu PheArg Thr 385 390 395 400 Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp MetGlu Asp Cys Gly 405 410 415 Tyr Asn Ile Pro Gln Thr Asp Glu Ser 420 42529 1362 DNA Homo sapiens 29 atggcccaag ccctgccctg gctcctgctg tggatgggcgcgggagtgct gcctgcccac 60 ggcacccagc acggcatccg gctgcccctg cgcagcggcctggggggcgc ccccctgggg 120 ctgcggctgc cccgggagac cgacgaagag cccgaggagcccggccggag gggcagcttt 180 gtggagatgg tggacaacct gaggggcaag tcggggcagggctactacgt ggagatgacc 240 gtgggcagcc ccccgcagac gctcaacatc ctggtggatacaggcagcag taactttgca 300 gtgggtgctg ccccccaccc cttcctgcat cgctactaccagaggcagct gtccagcaca 360 taccgggacc tccggaaggg tgtgtatgtg ccctacacccagggcaagtg ggaaggggag 420 ctgggcaccg acctggtaag catcccccat ggccccaacgtcactgtgcg tgccaacatt 480 gctgccatca ctgaatcaga caagttcttc atcaacggctccaactggga aggcatcctg 540 gggctggcct atgctgagat tgccaggcct gacgactccctggagccttt ctttgactct 600 ctggtaaagc agacccacgt tcccaacctc ttctccctgcacctttgtgg tgctggcttc 660 cccctcaacc agtctgaagt gctggcctct gtcggagggagcatgatcat tggaggtatc 720 gaccactcgc tgtacacagg cagtctctgg tatacacccatccggcggga gtggtattat 780 gaggtcatca ttgtgcgggt ggagatcaat ggacaggatctgaaaatgga ctgcaaggag 840 tacaactatg acaagagcat tgtggacagt ggcaccaccaaccttcgttt gcccaagaaa 900 gtgtttgaag ctgcagtcaa atccatcaag gcagcctcctccacggagaa gttccctgat 960 ggtttctggc taggagagca gctggtgtgc tggcaagcaggcaccacccc ttggaacatt 1020 ttcccagtca tctcactcta cctaatgggt gaggttaccaaccagtcctt ccgcatcacc 1080 atccttccgc agcaatacct gcggccagtg gaagatgtggccacgtccca agacgactgt 1140 tacaagtttg ccatctcaca gtcatccacg ggcactgttatgggagctgt tatcatggag 1200 ggcttctacg ttgtctttga tcgggcccga aaacgaattggctttgctgt cagcgcttgc 1260 catgtgcacg atgagttcag gacggcagcg gtggaaggcccttttgtcac cttggacatg 1320 gaagactgtg gctacaacat tccacagaca gatgagtcatga 1362 30 453 PRT Homo sapiens 30 Met Ala Gln Ala Leu Pro Trp Leu LeuLeu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly Thr Gln HisGly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Gly Gly Ala Pro Leu GlyLeu Arg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Pro Glu Glu Pro Gly ArgArg Gly Ser Phe Val Glu Met Val 50 55 60 Asp Asn Leu Arg Gly Lys Ser GlyGln Gly Tyr Tyr Val Glu Met Thr 65 70 75 80 Val Gly Ser Pro Pro Gln ThrLeu Asn Ile Leu Val Asp Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly AlaAla Pro His Pro Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu SerSer Thr Tyr Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr ThrGln Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser IlePro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala AlaIle Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 GluGly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Pro Asp Asp 180 185 190Ser Leu Glu Pro Phe Phe Asp Ser Leu Val Lys Gln Thr His Val Pro 195 200205 Asn Leu Phe Ser Leu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln 210215 220 Ser Glu Val Leu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile225 230 235 240 Asp His Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro IleArg Arg 245 250 255 Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu IleAsn Gly Gln 260 265 270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr AspLys Ser Ile Val 275 280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro LysLys Val Phe Glu Ala 290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser SerThr Glu Lys Phe Pro Asp 305 310 315 320 Gly Phe Trp Leu Gly Glu Gln LeuVal Cys Trp Gln Ala Gly Thr Thr 325 330 335 Pro Trp Asn Ile Phe Pro ValIle Ser Leu Tyr Leu Met Gly Glu Val 340 345 350 Thr Asn Gln Ser Phe ArgIle Thr Ile Leu Pro Gln Gln Tyr Leu Arg 355 360 365 Pro Val Glu Asp ValAla Thr Ser Gln Asp Asp Cys Tyr Lys Phe Ala 370 375 380 Ile Ser Gln SerSer Thr Gly Thr Val Met Gly Ala Val Ile Met Glu 385 390 395 400 Gly PheTyr Val Val Phe Asp Arg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 ValSer Ala Cys His Val His Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430Gly Pro Phe Val Thr Leu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440445 Gln Thr Asp Glu Ser 450 31 1380 DNA Homo sapiens 31 atggcccaagccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagcacggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120 ctgcggctgccccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180 gtggagatggtggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 240 gtgggcagccccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctgccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360 taccgggacctccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccgacctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatcactgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540 gggctggcctatgctgagat tgccaggcct gacgactccc tggagccttt ctttgactct 600 ctggtaaagcagacccacgt tcccaacctc ttctccctgc acctttgtgg tgctggcttc 660 cccctcaaccagtctgaagt gctggcctct gtcggaggga gcatgatcat tggaggtatc 720 gaccactcgctgtacacagg cagtctctgg tatacaccca tccggcggga gtggtattat 780 gaggtcatcattgtgcgggt ggagatcaat ggacaggatc tgaaaatgga ctgcaaggag 840 tacaactatgacaagagcat tgtggacagt ggcaccacca accttcgttt gcccaagaaa 900 gtgtttgaagctgcagtcaa atccatcaag gcagcctcct ccacggagaa gttccctgat 960 ggtttctggctaggagagca gctggtgtgc tggcaagcag gcaccacccc ttggaacatt 1020 ttcccagtcatctcactcta cctaatgggt gaggttacca accagtcctt ccgcatcacc 1080 atccttccgcagcaatacct gcggccagtg gaagatgtgg ccacgtccca agacgactgt 1140 tacaagtttgccatctcaca gtcatccacg ggcactgtta tgggagctgt tatcatggag 1200 ggcttctacgttgtctttga tcgggcccga aaacgaattg gctttgctgt cagcgcttgc 1260 catgtgcacgatgagttcag gacggcagcg gtggaaggcc cttttgtcac cttggacatg 1320 gaagactgtggctacaacat tccacagaca gatgagtcac agcagcagca gcagcagtga 1380 32 459 PRTHomo sapiens 32 Met Ala Gln Ala Leu Pro Trp Leu Leu Leu Trp Met Gly AlaGly Val 1 5 10 15 Leu Pro Ala His Gly Thr Gln His Gly Ile Arg Leu ProLeu Arg Ser 20 25 30 Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro ArgGlu Thr Asp 35 40 45 Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe ValGlu Met Val 50 55 60 Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr ValGlu Met Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu ValAsp Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro PheLeu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg AspLeu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr Gln Gly Lys Trp GluGly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile Pro His Gly Pro AsnVal Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala Ile Thr Glu Ser AspLys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 Glu Gly Ile Leu Gly LeuAla Tyr Ala Glu Ile Ala Arg Pro Asp Asp 180 185 190 Ser Leu Glu Pro PhePhe Asp Ser Leu Val Lys Gln Thr His Val Pro 195 200 205 Asn Leu Phe SerLeu Gln Leu Cys Gly Ala Gly Phe Pro Leu Asn Gln 210 215 220 Ser Glu ValLeu Ala Ser Val Gly Gly Ser Met Ile Ile Gly Gly Ile 225 230 235 240 AspHis Ser Leu Tyr Thr Gly Ser Leu Trp Tyr Thr Pro Ile Arg Arg 245 250 255Glu Trp Tyr Tyr Glu Val Ile Ile Val Arg Val Glu Ile Asn Gly Gln 260 265270 Asp Leu Lys Met Asp Cys Lys Glu Tyr Asn Tyr Asp Lys Ser Ile Val 275280 285 Asp Ser Gly Thr Thr Asn Leu Arg Leu Pro Lys Lys Val Phe Glu Ala290 295 300 Ala Val Lys Ser Ile Lys Ala Ala Ser Ser Thr Glu Lys Phe ProAsp 305 310 315 320 Gly Phe Trp Leu Gly Glu Gln Leu Val Cys Trp Gln AlaGly Thr Thr 325 330 335 Pro Trp Asn Ile Phe Pro Val Ile Ser Leu Tyr LeuMet Gly Glu Val 340 345 350 Thr Asn Gln Ser Phe Arg Ile Thr Ile Leu ProGln Gln Tyr Leu Arg 355 360 365 Pro Val Glu Asp Val Ala Thr Ser Gln AspAsp Cys Tyr Lys Phe Ala 370 375 380 Ile Ser Gln Ser Ser Thr Gly Thr ValMet Gly Ala Val Ile Met Glu 385 390 395 400 Gly Phe Tyr Val Val Phe AspArg Ala Arg Lys Arg Ile Gly Phe Ala 405 410 415 Val Ser Ala Cys His ValHis Asp Glu Phe Arg Thr Ala Ala Val Glu 420 425 430 Gly Pro Phe Val ThrLeu Asp Met Glu Asp Cys Gly Tyr Asn Ile Pro 435 440 445 Gln Thr Asp GluSer His His His His His His 450 455 33 25 PRT Homo sapiens 33 Ser GluGln Gln Arg Arg Pro Arg Asp Pro Glu Val Val Asn Asp Glu 1 5 10 15 SerSer Leu Val Arg His Arg Trp Lys 20 25 34 19 PRT Homo sapiens 34 Ser GluGln Leu Arg Gln Gln His Asp Asp Phe Ala Asp Asp Ile Ser 1 5 10 15 LeuLeu Lys 35 29 DNA Homo sapiens 35 gtggatccac ccagcacggc atccggctg 29 3636 DNA Homo sapiens 36 gaaagctttc atgactcatc tgtctgtgga atgttg 36 37 39DNA Homo sapiens 37 gatcgatgac tatctctgac tctccgcgtg aacaggacg 39 38 39DNA Homo sapiens 38 gatccgtcct gttcacgcgg agagtcagag atagtcatc 39 39 77DNA Artificial sequence Hu-Asp2 39 cggcatccgg ctgcccctgc gtagcggtctgggtggtgct ccactgggtc tgcgtctgcc 60 ccgggagacc gacgaag 77 40 77 DNAArtificial sequence Hu-Asp2 40 cttcgtcggt ctcccggggc agacgcagacccagtggagc accacccaga ccgctacgca 60 ggggcagccg gatgccg 77 41 51 DNACaspase-8 Cleavage Site 41 gatcgatgac tatctctgac tctccgctgg actctggtatcgaaaccgac g 51 42 51 DNA Caspase-8 Cleavage Site 42 gatccgtcggtttcgatacc agagtccagc ggagagtcag agatagtcat c 51 43 32 DNA Homo sapiens43 aaggatcctt tgtggagatg gtggacaacc tg 32 44 36 DNA Homo sapiens 44gaaagctttc atgactcatc tgtctgtgga atgttg 36 45 24 DNA 6-His tag 45gatcgcatca tcaccatcac catg 24 46 24 DNA 6-His tag 46 gatccatggtgatggtgatg atgc 24 47 22 DNA Artificial sequence Primer 47 gactgaccactcgaccaggt tc 22 48 51 DNA Artificial sequence Primer 48 cgaattaaattccagcacac tggctacttc ttgttctgca tctcaaagaa c 51 49 26 DNA Artificialsequence Primer 49 cgaattaaat tccagcacac tggcta 26 50 1287 DNAArtificial sequence Hu-Asp2(b) delta TM 50 atggcccaag ccctgccctggctcctgctg tggatgggcg cgggagtgct gcctgcccac 60 ggcacccagc acggcatccggctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120 ctgcggctgc cccgggagaccgacgaagag cccgaggagc ccggccggag gggcagcttt 180 gtggagatgg tggacaacctgaggggcaag tcggggcagg gctactacgt ggagatgacc 240 gtgggcagcc ccccgcagacgctcaacatc ctggtggata caggcagcag taactttgca 300 gtgggtgctg ccccccaccccttcctgcat cgctactacc agaggcagct gtccagcaca 360 taccgggacc tccggaagggtgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420 ctgggcaccg acctggtaagcatcccccat ggccccaacg tcactgtgcg tgccaacatt 480 gctgccatca ctgaatcagacaagttcttc atcaacggct ccaactggga aggcatcctg 540 gggctggcct atgctgagattgccaggctt tgtggtgctg gcttccccct caaccagtct 600 gaagtgctgg cctctgtcggagggagcatg atcattggag gtatcgacca ctcgctgtac 660 acaggcagtc tctggtatacacccatccgg cgggagtggt attatgaggt catcattgtg 720 cgggtggaga tcaatggacaggatctgaaa atggactgca aggagtacaa ctatgacaag 780 agcattgtgg acagtggcaccaccaacctt cgtttgccca agaaagtgtt tgaagctgca 840 gtcaaatcca tcaaggcagcctcctccacg gagaagttcc ctgatggttt ctggctagga 900 gagcagctgg tgtgctggcaagcaggcacc accccttgga acattttccc agtcatctca 960 ctctacctaa tgggtgaggttaccaaccag tccttccgca tcaccatcct tccgcagcaa 1020 tacctgcggc cagtggaagatgtggccacg tcccaagacg actgttacaa gtttgccatc 1080 tcacagtcat ccacgggcactgttatggga gctgttatca tggagggctt ctacgttgtc 1140 tttgatcggg cccgaaaacgaattggcttt gctgtcagcg cttgccatgt gcacgatgag 1200 ttcaggacgg cagcggtggaaggccctttt gtcaccttgg acatggaaga ctgtggctac 1260 aacattccac agacagatgagtcatga 1287 51 428 PRT Artificial sequence Hu-Asp2(b) delta TM 51 MetAla Gln Ala Leu Pro Trp Leu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15Leu Pro Ala His Gly Thr Gln His Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30Gly Leu Gly Gly Ala Pro Leu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45Glu Glu Pro Glu Glu Pro Gly Arg Arg Gly Ser Phe Val Glu Met Val 50 55 60Asp Asn Leu Arg Gly Lys Ser Gly Gln Gly Tyr Tyr Val Glu Met Thr 65 70 7580 Val Gly Ser Pro Pro Gln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 85 9095 Ser Asn Phe Ala Val Gly Ala Ala Pro His Pro Phe Leu His Arg Tyr 100105 110 Tyr Gln Arg Gln Leu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val115 120 125 Tyr Val Pro Tyr Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly ThrAsp 130 135 140 Leu Val Ser Ile Pro His Gly Pro Asn Val Thr Val Arg AlaAsn Ile 145 150 155 160 Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile AsnGly Ser Asn Trp 165 170 175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu IleAla Arg Leu Cys Gly 180 185 190 Ala Gly Phe Pro Leu Asn Gln Ser Glu ValLeu Ala Ser Val Gly Gly 195 200 205 Ser Met Ile Ile Gly Gly Ile Asp HisSer Leu Tyr Thr Gly Ser Leu 210 215 220 Trp Tyr Thr Pro Ile Arg Arg GluTrp Tyr Tyr Glu Val Ile Ile Val 225 230 235 240 Arg Val Glu Ile Asn GlyGln Asp Leu Lys Met Asp Cys Lys Glu Tyr 245 250 255 Asn Tyr Asp Lys SerIle Val Asp Ser Gly Thr Thr Asn Leu Arg Leu 260 265 270 Pro Lys Lys ValPhe Glu Ala Ala Val Lys Ser Ile Lys Ala Ala Ser 275 280 285 Ser Thr GluLys Phe Pro Asp Gly Phe Trp Leu Gly Glu Gln Leu Val 290 295 300 Cys TrpGln Ala Gly Thr Thr Pro Trp Asn Ile Phe Pro Val Ile Ser 305 310 315 320Leu Tyr Leu Met Gly Glu Val Thr Asn Gln Ser Phe Arg Ile Thr Ile 325 330335 Leu Pro Gln Gln Tyr Leu Arg Pro Val Glu Asp Val Ala Thr Ser Gln 340345 350 Asp Asp Cys Tyr Lys Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr Val355 360 365 Met Gly Ala Val Ile Met Glu Gly Phe Tyr Val Val Phe Asp ArgAla 370 375 380 Arg Lys Arg Ile Gly Phe Ala Val Ser Ala Cys His Val HisAsp Glu 385 390 395 400 Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val ThrLeu Asp Met Glu 405 410 415 Asp Cys Gly Tyr Asn Ile Pro Gln Thr Asp GluSer 420 425 52 1305 DNA Artificial sequence Hu-Asp2(b) delta TM 52atggcccaag ccctgccctg gctcctgctg tggatgggcg cgggagtgct gcctgcccac 60ggcacccagc acggcatccg gctgcccctg cgcagcggcc tggggggcgc ccccctgggg 120ctgcggctgc cccgggagac cgacgaagag cccgaggagc ccggccggag gggcagcttt 180gtggagatgg tggacaacct gaggggcaag tcggggcagg gctactacgt ggagatgacc 240gtgggcagcc ccccgcagac gctcaacatc ctggtggata caggcagcag taactttgca 300gtgggtgctg ccccccaccc cttcctgcat cgctactacc agaggcagct gtccagcaca 360taccgggacc tccggaaggg tgtgtatgtg ccctacaccc agggcaagtg ggaaggggag 420ctgggcaccg acctggtaag catcccccat ggccccaacg tcactgtgcg tgccaacatt 480gctgccatca ctgaatcaga caagttcttc atcaacggct ccaactggga aggcatcctg 540gggctggcct atgctgagat tgccaggctt tgtggtgctg gcttccccct caaccagtct 600gaagtgctgg cctctgtcgg agggagcatg atcattggag gtatcgacca ctcgctgtac 660acaggcagtc tctggtatac acccatccgg cgggagtggt attatgaggt catcattgtg 720cgggtggaga tcaatggaca ggatctgaaa atggactgca aggagtacaa ctatgacaag 780agcattgtgg acagtggcac caccaacctt cgtttgccca agaaagtgtt tgaagctgca 840gtcaaatcca tcaaggcagc ctcctccacg gagaagttcc ctgatggttt ctggctagga 900gagcagctgg tgtgctggca agcaggcacc accccttgga acattttccc agtcatctca 960ctctacctaa tgggtgaggt taccaaccag tccttccgca tcaccatcct tccgcagcaa 1020tacctgcggc cagtggaaga tgtggccacg tcccaagacg actgttacaa gtttgccatc 1080tcacagtcat ccacgggcac tgttatggga gctgttatca tggagggctt ctacgttgtc 1140tttgatcggg cccgaaaacg aattggcttt gctgtcagcg cttgccatgt gcacgatgag 1200ttcaggacgg cagcggtgga aggccctttt gtcaccttgg acatggaaga ctgtggctac 1260aacattccac agacagatga gtcacagcag cagcagcagc agtga 1305 53 434 PRTArtificial sequence Hu-Asp2(b) delta TM 53 Met Ala Gln Ala Leu Pro TrpLeu Leu Leu Trp Met Gly Ala Gly Val 1 5 10 15 Leu Pro Ala His Gly ThrGln His Gly Ile Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Gly Gly Ala ProLeu Gly Leu Arg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Pro Glu Glu ProGly Arg Arg Gly Ser Phe Val Glu Met Val 50 55 60 Asp Asn Leu Arg Gly LysSer Gly Gln Gly Tyr Tyr Val Glu Met Thr 65 70 75 80 Val Gly Ser Pro ProGln Thr Leu Asn Ile Leu Val Asp Thr Gly Ser 85 90 95 Ser Asn Phe Ala ValGly Ala Ala Pro His Pro Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg GlnLeu Ser Ser Thr Tyr Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val ProTyr Thr Gln Gly Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu ValSer Ile Pro His Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160Ala Ala Ile Thr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170175 Glu Gly Ile Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 180185 190 Ala Gly Phe Pro Leu Asn Gln Ser Glu Val Leu Ala Ser Val Gly Gly195 200 205 Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly SerLeu 210 215 220 Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val IleIle Val 225 230 235 240 Arg Val Glu Ile Asn Gly Gln Asp Leu Lys Met AspCys Lys Glu Tyr 245 250 255 Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly ThrThr Asn Leu Arg Leu 260 265 270 Pro Lys Lys Val Phe Glu Ala Ala Val LysSer Ile Lys Ala Ala Ser 275 280 285 Ser Thr Glu Lys Phe Pro Asp Gly PheTrp Leu Gly Glu Gln Leu Val 290 295 300 Cys Trp Gln Ala Gly Thr Thr ProTrp Asn Ile Phe Pro Val Ile Ser 305 310 315 320 Leu Tyr Leu Met Gly GluVal Thr Asn Gln Ser Phe Arg Ile Thr Ile 325 330 335 Leu Pro Gln Gln TyrLeu Arg Pro Val Glu Asp Val Ala Thr Ser Gln 340 345 350 Asp Asp Cys TyrLys Phe Ala Ile Ser Gln Ser Ser Thr Gly Thr Val 355 360 365 Met Gly AlaVal Ile Met Glu Gly Phe Tyr Val Val Phe Asp Arg Ala 370 375 380 Arg LysArg Ile Gly Phe Ala Val Ser Ala Cys His Val His Asp Glu 385 390 395 400Phe Arg Thr Ala Ala Val Glu Gly Pro Phe Val Thr Leu Asp Met Glu 405 410415 Asp Cys Gly Tyr Asn Ile Pro Gln Thr Asp Glu Ser His His His His 420425 430 His His 54 2310 DNA Homo sapiens 54 atgctgcccg gtttggcactgctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatg gtaatgctggcctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgc acatgaatgtccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattg ataccaaggaaggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcacca atgtggtagaagccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagc agtgcaagacccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaa gtgatgcccttctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgttt gcgaaactcatcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtacca acttgcatgactacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagt ttgtgtgttgcccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggagg atgactcggatgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagaca aagtagtagaagtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatg atgacgaggacgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaag aagccacagagagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtgg aagaggtggttcgagaggtg tgctctgaac aagccgagac ggggccgtgc 900 cgagcaatga tctcccgctggtactttgat gtgactgaag ggaagtgtgc cccattcttt 960 tacggcggat gtggcggcaaccggaacaac tttgacacag aagagtactg catggccgtg 1020 tgtggcagcg ccatgtcccaaagtttactc aagactaccc aggaacctct tggccgagat 1080 cctgttaaac ttcctacaacagcagccagt acccctgatg ccgttgacaa gtatctcgag 1140 acacctgggg atgagaatgaacatgcccat ttccagaaag ccaaagagag gcttgaggcc 1200 aagcaccgag agagaatgtcccaggtcatg agagaatggg aagaggcaga acgtcaagca 1260 aagaacttgc ctaaagctgataagaaggca gttatccagc atttccagga gaaagtggaa 1320 tctttggaac aggaagcagccaacgagaga cagcagctgg tggagacaca catggccaga 1380 gtggaagcca tgctcaatgaccgccgccgc ctggccctgg agaactacat caccgctctg 1440 caggctgttc ctcctcggcctcgtcacgtg ttcaatatgc taaagaagta tgtccgcgca 1500 gaacagaagg acagacagcacaccctaaag catttcgagc atgtgcgcat ggtggatccc 1560 aagaaagccg ctcagatccggtcccaggtt atgacacacc tccgtgtgat ttatgagcgc 1620 atgaatcagt ctctctccctgctctacaac gtgcctgcag tggccgagga gattcaggat 1680 gaagttgatg agctgcttcagaaagagcaa aactattcag atgacgtctt ggccaacatg 1740 attagtgaac caaggatcagttacggaaac gatgctctca tgccatcttt gaccgaaacg 1800 aaaaccaccg tggagctccttcccgtgaat ggagagttca gcctggacga tctccagccg 1860 tggcattctt ttggggctgactctgtgcca gccaacacag aaaacgaagt tgagcctgtt 1920 gatgcccgcc ctgctgccgaccgaggactg accactcgac caggttctgg gttgacaaat 1980 atcaagacgg aggagatctctgaagtgaag atggatgcag aattccgaca tgactcagga 2040 tatgaagttc atcatcaaaaattggtgttc tttgcagaag atgtgggttc aaacaaaggt 2100 gcaatcattg gactcatggtgggcggtgtt gtcatagcga cagtgatcgt catcaccttg 2160 gtgatgctga agaagaaacagtacacatcc attcatcatg gtgtggtgga ggttgacgcc 2220 gctgtcaccc cagaggagcgccacctgtcc aagatgcagc agaacggcta cgaaaatcca 2280 acctacaagt tctttgagcagatgcagaac 2310 55 770 PRT Homo sapiens 55 Met Leu Pro Gly Leu Ala LeuLeu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 Ala Leu Glu Val Pro ThrAsp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 Gln Ile Ala Met Phe CysGly Arg Leu Asn Met His Met Asn Val Gln 35 40 45 Asn Gly Lys Trp Asp SerAsp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 Thr Lys Glu Gly Ile LeuGln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80 Gln Ile Thr Asn ValVal Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95 Trp Cys Lys Arg GlyArg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105 110 Ile Pro Tyr ArgCys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115 120 125 Val Pro AspLys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys 130 135 140 Glu ThrHis Leu His Trp His Thr Val Ala Lys Glu Thr Cys Ser Glu 145 150 155 160Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu Pro Cys Gly Ile 165 170175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys Pro Leu Ala Glu Glu 180185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu Glu Asp Asp Ser Asp Val195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr Ala Asp Gly Ser Glu AspLys 210 215 220 Val Val Glu Val Ala Glu Glu Glu Glu Val Ala Glu Val GluGlu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu Asp Asp Glu Asp Gly AspGlu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr GluArg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr Thr Thr Thr Thr Glu SerVal Glu Glu Val Val Arg 275 280 285 Glu Val Cys Ser Glu Gln Ala Glu ThrGly Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg Trp Tyr Phe Asp Val ThrGlu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 Tyr Gly Gly Cys Gly GlyAsn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335 Cys Met Ala Val CysGly Ser Ala Met Ser Gln Ser Leu Leu Lys Thr 340 345 350 Thr Gln Glu ProLeu Ala Arg Asp Pro Val Lys Leu Pro Thr Thr Ala 355 360 365 Ala Ser ThrPro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp 370 375 380 Glu AsnGlu His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala 385 390 395 400Lys His Arg Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala 405 410415 Glu Arg Gln Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val Ile 420425 430 Gln His Phe Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn435 440 445 Glu Arg Gln Gln Leu Val Glu Thr His Met Ala Arg Val Glu AlaMet 450 455 460 Leu Asn Asp Arg Arg Arg Leu Ala Leu Glu Asn Tyr Ile ThrAla Leu 465 470 475 480 Gln Ala Val Pro Pro Arg Pro Arg His Val Phe AsnMet Leu Lys Lys 485 490 495 Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln HisThr Leu Lys His Phe 500 505 510 Glu His Val Arg Met Val Asp Pro Lys LysAla Ala Gln Ile Arg Ser 515 520 525 Gln Val Met Thr His Leu Arg Val IleTyr Glu Arg Met Asn Gln Ser 530 535 540 Leu Ser Leu Leu Tyr Asn Val ProAla Val Ala Glu Glu Ile Gln Asp 545 550 555 560 Glu Val Asp Glu Leu LeuGln Lys Glu Gln Asn Tyr Ser Asp Asp Val 565 570 575 Leu Ala Asn Met IleSer Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala 580 585 590 Leu Met Pro SerLeu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro 595 600 605 Val Asn GlyGlu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe 610 615 620 Gly AlaAsp Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val 625 630 635 640Asp Ala Arg Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser 645 650655 Gly Leu Thr Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met Asp 660665 670 Ala Glu Phe Arg His Asp Ser Gly Tyr Glu Val His His Gln Lys Leu675 680 685 Val Phe Phe Ala Glu Asp Val Gly Ser Asn Lys Gly Ala Ile IleGly 690 695 700 Leu Met Val Gly Gly Val Val Ile Ala Thr Val Ile Val IleThr Leu 705 710 715 720 Val Met Leu Lys Lys Lys Gln Tyr Thr Ser Ile HisHis Gly Val Val 725 730 735 Glu Val Asp Ala Ala Val Thr Pro Glu Glu ArgHis Leu Ser Lys Met 740 745 750 Gln Gln Asn Gly Tyr Glu Asn Pro Thr TyrLys Phe Phe Glu Gln Met 755 760 765 Gln Asn 770 56 2253 DNA Homo sapiens56 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac aagccgagac ggggccgtgc 900cgagcaatga tctcccgctg gtactttgat gtgactgaag ggaagtgtgc cccattcttt 960tacggcggat gtggcggcaa ccggaacaac tttgacacag aagagtactg catggccgtg 1020tgtggcagcg ccattcctac aacagcagcc agtacccctg atgccgttga caagtatctc 1080gagacacctg gggatgagaa tgaacatgcc catttccaga aagccaaaga gaggcttgag 1140gccaagcacc gagagagaat gtcccaggtc atgagagaat gggaagaggc agaacgtcaa 1200gcaaagaact tgcctaaagc tgataagaag gcagttatcc agcatttcca ggagaaagtg 1260gaatctttgg aacaggaagc agccaacgag agacagcagc tggtggagac acacatggcc 1320agagtggaag ccatgctcaa tgaccgccgc cgcctggccc tggagaacta catcaccgct 1380ctgcaggctg ttcctcctcg gcctcgtcac gtgttcaata tgctaaagaa gtatgtccgc 1440gcagaacaga aggacagaca gcacacccta aagcatttcg agcatgtgcg catggtggat 1500cccaagaaag ccgctcagat ccggtcccag gttatgacac acctccgtgt gatttatgag 1560cgcatgaatc agtctctctc cctgctctac aacgtgcctg cagtggccga ggagattcag 1620gatgaagttg atgagctgct tcagaaagag caaaactatt cagatgacgt cttggccaac 1680atgattagtg aaccaaggat cagttacgga aacgatgctc tcatgccatc tttgaccgaa 1740acgaaaacca ccgtggagct ccttcccgtg aatggagagt tcagcctgga cgatctccag 1800ccgtggcatt cttttggggc tgactctgtg ccagccaaca cagaaaacga agttgagcct 1860gttgatgccc gccctgctgc cgaccgagga ctgaccactc gaccaggttc tgggttgaca 1920aatatcaaga cggaggagat ctctgaagtg aagatggatg cagaattccg acatgactca 1980ggatatgaag ttcatcatca aaaattggtg ttctttgcag aagatgtggg ttcaaacaaa 2040ggtgcaatca ttggactcat ggtgggcggt gttgtcatag cgacagtgat cgtcatcacc 2100ttggtgatgc tgaagaagaa acagtacaca tccattcatc atggtgtggt ggaggttgac 2160gccgctgtca ccccagagga gcgccacctg tccaagatgc agcagaacgg ctacgaaaat 2220ccaacctaca agttctttga gcagatgcag aac 2253 57 751 PRT Homo sapiens 57 MetLeu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 7580 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 9095 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp ValCys 130 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr CysSer Glu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu LeuPro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys CysPro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala GluGlu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp TyrAla Asp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu GluGlu Val Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp GluAsp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu ProTyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr ThrThr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Glu Val CysSer Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 Ser ArgTrp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330335 Cys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340345 350 Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu355 360 365 His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys HisArg 370 375 380 Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala GluArg Gln 385 390 395 400 Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala ValIle Gln His Phe 405 410 415 Gln Glu Lys Val Glu Ser Leu Glu Gln Glu AlaAla Asn Glu Arg Gln 420 425 430 Gln Leu Val Glu Thr His Met Ala Arg ValGlu Ala Met Leu Asn Asp 435 440 445 Arg Arg Arg Leu Ala Leu Glu Asn TyrIle Thr Ala Leu Gln Ala Val 450 455 460 Pro Pro Arg Pro Arg His Val PheAsn Met Leu Lys Lys Tyr Val Arg 465 470 475 480 Ala Glu Gln Lys Asp ArgGln His Thr Leu Lys His Phe Glu His Val 485 490 495 Arg Met Val Asp ProLys Lys Ala Ala Gln Ile Arg Ser Gln Val Met 500 505 510 Thr His Leu ArgVal Ile Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu 515 520 525 Leu Tyr AsnVal Pro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp 530 535 540 Glu LeuLeu Gln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560Met Ile Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570575 Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580585 590 Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp595 600 605 Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp AlaArg 610 615 620 Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser GlyLeu Thr 625 630 635 640 Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys MetAsp Ala Glu Phe 645 650 655 Arg His Asp Ser Gly Tyr Glu Val His His GlnLys Leu Val Phe Phe 660 665 670 Ala Glu Asp Val Gly Ser Asn Lys Gly AlaIle Ile Gly Leu Met Val 675 680 685 Gly Gly Val Val Ile Ala Thr Val IleVal Ile Thr Leu Val Met Leu 690 695 700 Lys Lys Lys Gln Tyr Thr Ser IleHis His Gly Val Val Glu Val Asp 705 710 715 720 Ala Ala Val Thr Pro GluGlu Arg His Leu Ser Lys Met Gln Gln Asn 725 730 735 Gly Tyr Glu Asn ProThr Tyr Lys Phe Phe Glu Gln Met Gln Asn 740 745 750 58 2316 DNA Homosapiens 58 atgctgcccg gtttggcact gctcctgctg gccgcctgga cggctcgggcgctggaggta 60 cccactgatg gtaatgctgg cctgctggct gaaccccaga ttgccatgttctgtggcaga 120 ctgaacatgc acatgaatgt ccagaatggg aagtgggatt cagatccatcagggaccaaa 180 acctgcattg ataccaagga aggcatcctg cagtattgcc aagaagtctaccctgaactg 240 cagatcacca atgtggtaga agccaaccaa ccagtgacca tccagaactggtgcaagcgg 300 ggccgcaagc agtgcaagac ccatccccac tttgtgattc cctaccgctgcttagttggt 360 gagtttgtaa gtgatgccct tctcgttcct gacaagtgca aattcttacaccaggagagg 420 atggatgttt gcgaaactca tcttcactgg cacaccgtcg ccaaagagacatgcagtgag 480 aagagtacca acttgcatga ctacggcatg ttgctgccct gcggaattgacaagttccga 540 ggggtagagt ttgtgtgttg cccactggct gaagaaagtg acaatgtggattctgctgat 600 gcggaggagg atgactcgga tgtctggtgg ggcggagcag acacagactatgcagatggg 660 agtgaagaca aagtagtaga agtagcagag gaggaagaag tggctgaggtggaagaagaa 720 gaagccgatg atgacgagga cgatgaggat ggtgatgagg tagaggaagaggctgaggaa 780 ccctacgaag aagccacaga gagaaccacc agcattgcca ccaccaccaccaccaccaca 840 gagtctgtgg aagaggtggt tcgagaggtg tgctctgaac aagccgagacggggccgtgc 900 cgagcaatga tctcccgctg gtactttgat gtgactgaag ggaagtgtgccccattcttt 960 tacggcggat gtggcggcaa ccggaacaac tttgacacag aagagtactgcatggccgtg 1020 tgtggcagcg ccatgtccca aagtttactc aagactaccc aggaacctcttggccgagat 1080 cctgttaaac ttcctacaac agcagccagt acccctgatg ccgttgacaagtatctcgag 1140 acacctgggg atgagaatga acatgcccat ttccagaaag ccaaagagaggcttgaggcc 1200 aagcaccgag agagaatgtc ccaggtcatg agagaatggg aagaggcagaacgtcaagca 1260 aagaacttgc ctaaagctga taagaaggca gttatccagc atttccaggagaaagtggaa 1320 tctttggaac aggaagcagc caacgagaga cagcagctgg tggagacacacatggccaga 1380 gtggaagcca tgctcaatga ccgccgccgc ctggccctgg agaactacatcaccgctctg 1440 caggctgttc ctcctcggcc tcgtcacgtg ttcaatatgc taaagaagtatgtccgcgca 1500 gaacagaagg acagacagca caccctaaag catttcgagc atgtgcgcatggtggatccc 1560 aagaaagccg ctcagatccg gtcccaggtt atgacacacc tccgtgtgatttatgagcgc 1620 atgaatcagt ctctctccct gctctacaac gtgcctgcag tggccgaggagattcaggat 1680 gaagttgatg agctgcttca gaaagagcaa aactattcag atgacgtcttggccaacatg 1740 attagtgaac caaggatcag ttacggaaac gatgctctca tgccatctttgaccgaaacg 1800 aaaaccaccg tggagctcct tcccgtgaat ggagagttca gcctggacgatctccagccg 1860 tggcattctt ttggggctga ctctgtgcca gccaacacag aaaacgaagttgagcctgtt 1920 gatgcccgcc ctgctgccga ccgaggactg accactcgac caggttctgggttgacaaat 1980 atcaagacgg aggagatctc tgaagtgaag atggatgcag aattccgacatgactcagga 2040 tatgaagttc atcatcaaaa attggtgttc tttgcagaag atgtgggttcaaacaaaggt 2100 gcaatcattg gactcatggt gggcggtgtt gtcatagcga cagtgatcgtcatcaccttg 2160 gtgatgctga agaagaaaca gtacacatcc attcatcatg gtgtggtggaggttgacgcc 2220 gctgtcaccc cagaggagcg ccacctgtcc aagatgcagc agaacggctacgaaaatcca 2280 acctacaagt tctttgagca gatgcagaac aagaag 2316 59 772 PRTHomo sapiens 59 Met Leu Pro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp ThrAla Arg 1 5 10 15 Ala Leu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu LeuAla Glu Pro 20 25 30 Gln Ile Ala Met Phe Cys Gly Arg Leu Asn Met His MetAsn Val Gln 35 40 45 Asn Gly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys ThrCys Ile Asp 50 55 60 Thr Lys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val TyrPro Glu Leu 65 70 75 80 Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro ValThr Ile Gln Asn 85 90 95 Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr HisPro His Phe Val 100 105 110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe ValSer Asp Ala Leu Leu 115 120 125 Val Pro Asp Lys Cys Lys Phe Leu His GlnGlu Arg Met Asp Val Cys 130 135 140 Glu Thr His Leu His Trp His Thr ValAla Lys Glu Thr Cys Ser Glu 145 150 155 160 Lys Ser Thr Asn Leu His AspTyr Gly Met Leu Leu Pro Cys Gly Ile 165 170 175 Asp Lys Phe Arg Gly ValGlu Phe Val Cys Cys Pro Leu Ala Glu Glu 180 185 190 Ser Asp Asn Val AspSer Ala Asp Ala Glu Glu Asp Asp Ser Asp Val 195 200 205 Trp Trp Gly GlyAla Asp Thr Asp Tyr Ala Asp Gly Ser Glu Asp Lys 210 215 220 Val Val GluVal Ala Glu Glu Glu Glu Val Ala Glu Val Glu Glu Glu 225 230 235 240 GluAla Asp Asp Asp Glu Asp Asp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255Glu Ala Glu Glu Pro Tyr Glu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265270 Ala Thr Thr Thr Thr Thr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275280 285 Glu Val Cys Ser Glu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile290 295 300 Ser Arg Trp Tyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro PhePhe 305 310 315 320 Tyr Gly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp ThrGlu Glu Tyr 325 330 335 Cys Met Ala Val Cys Gly Ser Ala Met Ser Gln SerLeu Leu Lys Thr 340 345 350 Thr Gln Glu Pro Leu Ala Arg Asp Pro Val LysLeu Pro Thr Thr Ala 355 360 365 Ala Ser Thr Pro Asp Ala Val Asp Lys TyrLeu Glu Thr Pro Gly Asp 370 375 380 Glu Asn Glu His Ala His Phe Gln LysAla Lys Glu Arg Leu Glu Ala 385 390 395 400 Lys His Arg Glu Arg Met SerGln Val Met Arg Glu Trp Glu Glu Ala 405 410 415 Glu Arg Gln Ala Lys AsnLeu Pro Lys Ala Asp Lys Lys Ala Val Ile 420 425 430 Gln His Phe Gln GluLys Val Glu Ser Leu Glu Gln Glu Ala Ala Asn 435 440 445 Glu Arg Gln GlnLeu Val Glu Thr His Met Ala Arg Val Glu Ala Met 450 455 460 Leu Asn AspArg Arg Arg Leu Ala Leu Glu Asn Tyr Ile Thr Ala Leu 465 470 475 480 GlnAla Val Pro Pro Arg Pro Arg His Val Phe Asn Met Leu Lys Lys 485 490 495Tyr Val Arg Ala Glu Gln Lys Asp Arg Gln His Thr Leu Lys His Phe 500 505510 Glu His Val Arg Met Val Asp Pro Lys Lys Ala Ala Gln Ile Arg Ser 515520 525 Gln Val Met Thr His Leu Arg Val Ile Tyr Glu Arg Met Asn Gln Ser530 535 540 Leu Ser Leu Leu Tyr Asn Val Pro Ala Val Ala Glu Glu Ile GlnAsp 545 550 555 560 Glu Val Asp Glu Leu Leu Gln Lys Glu Gln Asn Tyr SerAsp Asp Val 565 570 575 Leu Ala Asn Met Ile Ser Glu Pro Arg Ile Ser TyrGly Asn Asp Ala 580 585 590 Leu Met Pro Ser Leu Thr Glu Thr Lys Thr ThrVal Glu Leu Leu Pro 595 600 605 Val Asn Gly Glu Phe Ser Leu Asp Asp LeuGln Pro Trp His Ser Phe 610 615 620 Gly Ala Asp Ser Val Pro Ala Asn ThrGlu Asn Glu Val Glu Pro Val 625 630 635 640 Asp Ala Arg Pro Ala Ala AspArg Gly Leu Thr Thr Arg Pro Gly Ser 645 650 655 Gly Leu Thr Asn Ile LysThr Glu Glu Ile Ser Glu Val Lys Met Asp 660 665 670 Ala Glu Phe Arg HisAsp Ser Gly Tyr Glu Val His His Gln Lys Leu 675 680 685 Val Phe Phe AlaGlu Asp Val Gly Ser Asn Lys Gly Ala Ile Ile Gly 690 695 700 Leu Met ValGly Gly Val Val Ile Ala Thr Val Ile Val Ile Thr Leu 705 710 715 720 ValMet Leu Lys Lys Lys Gln Tyr Thr Ser Ile His His Gly Val Val 725 730 735Glu Val Asp Ala Ala Val Thr Pro Glu Glu Arg His Leu Ser Lys Met 740 745750 Gln Gln Asn Gly Tyr Glu Asn Pro Thr Tyr Lys Phe Phe Glu Gln Met 755760 765 Gln Asn Lys Lys 770 60 2259 DNA Homo sapiens 60 atgctgcccggtttggcact gctcctgctg gccgcctgga cggctcgggc gctggaggta 60 cccactgatggtaatgctgg cctgctggct gaaccccaga ttgccatgtt ctgtggcaga 120 ctgaacatgcacatgaatgt ccagaatggg aagtgggatt cagatccatc agggaccaaa 180 acctgcattgataccaagga aggcatcctg cagtattgcc aagaagtcta ccctgaactg 240 cagatcaccaatgtggtaga agccaaccaa ccagtgacca tccagaactg gtgcaagcgg 300 ggccgcaagcagtgcaagac ccatccccac tttgtgattc cctaccgctg cttagttggt 360 gagtttgtaagtgatgccct tctcgttcct gacaagtgca aattcttaca ccaggagagg 420 atggatgtttgcgaaactca tcttcactgg cacaccgtcg ccaaagagac atgcagtgag 480 aagagtaccaacttgcatga ctacggcatg ttgctgccct gcggaattga caagttccga 540 ggggtagagtttgtgtgttg cccactggct gaagaaagtg acaatgtgga ttctgctgat 600 gcggaggaggatgactcgga tgtctggtgg ggcggagcag acacagacta tgcagatggg 660 agtgaagacaaagtagtaga agtagcagag gaggaagaag tggctgaggt ggaagaagaa 720 gaagccgatgatgacgagga cgatgaggat ggtgatgagg tagaggaaga ggctgaggaa 780 ccctacgaagaagccacaga gagaaccacc agcattgcca ccaccaccac caccaccaca 840 gagtctgtggaagaggtggt tcgagaggtg tgctctgaac aagccgagac ggggccgtgc 900 cgagcaatgatctcccgctg gtactttgat gtgactgaag ggaagtgtgc cccattcttt 960 tacggcggatgtggcggcaa ccggaacaac tttgacacag aagagtactg catggccgtg 1020 tgtggcagcgccattcctac aacagcagcc agtacccctg atgccgttga caagtatctc 1080 gagacacctggggatgagaa tgaacatgcc catttccaga aagccaaaga gaggcttgag 1140 gccaagcaccgagagagaat gtcccaggtc atgagagaat gggaagaggc agaacgtcaa 1200 gcaaagaacttgcctaaagc tgataagaag gcagttatcc agcatttcca ggagaaagtg 1260 gaatctttggaacaggaagc agccaacgag agacagcagc tggtggagac acacatggcc 1320 agagtggaagccatgctcaa tgaccgccgc cgcctggccc tggagaacta catcaccgct 1380 ctgcaggctgttcctcctcg gcctcgtcac gtgttcaata tgctaaagaa gtatgtccgc 1440 gcagaacagaaggacagaca gcacacccta aagcatttcg agcatgtgcg catggtggat 1500 cccaagaaagccgctcagat ccggtcccag gttatgacac acctccgtgt gatttatgag 1560 cgcatgaatcagtctctctc cctgctctac aacgtgcctg cagtggccga ggagattcag 1620 gatgaagttgatgagctgct tcagaaagag caaaactatt cagatgacgt cttggccaac 1680 atgattagtgaaccaaggat cagttacgga aacgatgctc tcatgccatc tttgaccgaa 1740 acgaaaaccaccgtggagct ccttcccgtg aatggagagt tcagcctgga cgatctccag 1800 ccgtggcattcttttggggc tgactctgtg ccagccaaca cagaaaacga agttgagcct 1860 gttgatgcccgccctgctgc cgaccgagga ctgaccactc gaccaggttc tgggttgaca 1920 aatatcaagacggaggagat ctctgaagtg aagatggatg cagaattccg acatgactca 1980 ggatatgaagttcatcatca aaaattggtg ttctttgcag aagatgtggg ttcaaacaaa 2040 ggtgcaatcattggactcat ggtgggcggt gttgtcatag cgacagtgat cgtcatcacc 2100 ttggtgatgctgaagaagaa acagtacaca tccattcatc atggtgtggt ggaggttgac 2160 gccgctgtcaccccagagga gcgccacctg tccaagatgc agcagaacgg ctacgaaaat 2220 ccaacctacaagttctttga gcagatgcag aacaagaag 2259 61 753 PRT Homo sapiens 61 Met LeuPro Gly Leu Ala Leu Leu Leu Leu Ala Ala Trp Thr Ala Arg 1 5 10 15 AlaLeu Glu Val Pro Thr Asp Gly Asn Ala Gly Leu Leu Ala Glu Pro 20 25 30 GlnIle Ala Met Phe Cys Gly Arg Leu Asn Met His Met Asn Val Gln 35 40 45 AsnGly Lys Trp Asp Ser Asp Pro Ser Gly Thr Lys Thr Cys Ile Asp 50 55 60 ThrLys Glu Gly Ile Leu Gln Tyr Cys Gln Glu Val Tyr Pro Glu Leu 65 70 75 80Gln Ile Thr Asn Val Val Glu Ala Asn Gln Pro Val Thr Ile Gln Asn 85 90 95Trp Cys Lys Arg Gly Arg Lys Gln Cys Lys Thr His Pro His Phe Val 100 105110 Ile Pro Tyr Arg Cys Leu Val Gly Glu Phe Val Ser Asp Ala Leu Leu 115120 125 Val Pro Asp Lys Cys Lys Phe Leu His Gln Glu Arg Met Asp Val Cys130 135 140 Glu Thr His Leu His Trp His Thr Val Ala Lys Glu Thr Cys SerGlu 145 150 155 160 Lys Ser Thr Asn Leu His Asp Tyr Gly Met Leu Leu ProCys Gly Ile 165 170 175 Asp Lys Phe Arg Gly Val Glu Phe Val Cys Cys ProLeu Ala Glu Glu 180 185 190 Ser Asp Asn Val Asp Ser Ala Asp Ala Glu GluAsp Asp Ser Asp Val 195 200 205 Trp Trp Gly Gly Ala Asp Thr Asp Tyr AlaAsp Gly Ser Glu Asp Lys 210 215 220 Val Val Glu Val Ala Glu Glu Glu GluVal Ala Glu Val Glu Glu Glu 225 230 235 240 Glu Ala Asp Asp Asp Glu AspAsp Glu Asp Gly Asp Glu Val Glu Glu 245 250 255 Glu Ala Glu Glu Pro TyrGlu Glu Ala Thr Glu Arg Thr Thr Ser Ile 260 265 270 Ala Thr Thr Thr ThrThr Thr Thr Glu Ser Val Glu Glu Val Val Arg 275 280 285 Glu Val Cys SerGlu Gln Ala Glu Thr Gly Pro Cys Arg Ala Met Ile 290 295 300 Ser Arg TrpTyr Phe Asp Val Thr Glu Gly Lys Cys Ala Pro Phe Phe 305 310 315 320 TyrGly Gly Cys Gly Gly Asn Arg Asn Asn Phe Asp Thr Glu Glu Tyr 325 330 335Cys Met Ala Val Cys Gly Ser Ala Ile Pro Thr Thr Ala Ala Ser Thr 340 345350 Pro Asp Ala Val Asp Lys Tyr Leu Glu Thr Pro Gly Asp Glu Asn Glu 355360 365 His Ala His Phe Gln Lys Ala Lys Glu Arg Leu Glu Ala Lys His Arg370 375 380 Glu Arg Met Ser Gln Val Met Arg Glu Trp Glu Glu Ala Glu ArgGln 385 390 395 400 Ala Lys Asn Leu Pro Lys Ala Asp Lys Lys Ala Val IleGln His Phe 405 410 415 Gln Glu Lys Val Glu Ser Leu Glu Gln Glu Ala AlaAsn Glu Arg Gln 420 425 430 Gln Leu Val Glu Thr His Met Ala Arg Val GluAla Met Leu Asn Asp 435 440 445 Arg Arg Arg Leu Ala Leu Glu Asn Tyr IleThr Ala Leu Gln Ala Val 450 455 460 Pro Pro Arg Pro Arg His Val Phe AsnMet Leu Lys Lys Tyr Val Arg 465 470 475 480 Ala Glu Gln Lys Asp Arg GlnHis Thr Leu Lys His Phe Glu His Val 485 490 495 Arg Met Val Asp Pro LysLys Ala Ala Gln Ile Arg Ser Gln Val Met 500 505 510 Thr His Leu Arg ValIle Tyr Glu Arg Met Asn Gln Ser Leu Ser Leu 515 520 525 Leu Tyr Asn ValPro Ala Val Ala Glu Glu Ile Gln Asp Glu Val Asp 530 535 540 Glu Leu LeuGln Lys Glu Gln Asn Tyr Ser Asp Asp Val Leu Ala Asn 545 550 555 560 MetIle Ser Glu Pro Arg Ile Ser Tyr Gly Asn Asp Ala Leu Met Pro 565 570 575Ser Leu Thr Glu Thr Lys Thr Thr Val Glu Leu Leu Pro Val Asn Gly 580 585590 Glu Phe Ser Leu Asp Asp Leu Gln Pro Trp His Ser Phe Gly Ala Asp 595600 605 Ser Val Pro Ala Asn Thr Glu Asn Glu Val Glu Pro Val Asp Ala Arg610 615 620 Pro Ala Ala Asp Arg Gly Leu Thr Thr Arg Pro Gly Ser Gly LeuThr 625 630 635 640 Asn Ile Lys Thr Glu Glu Ile Ser Glu Val Lys Met AspAla Glu Phe 645 650 655 Arg His Asp Ser Gly Tyr Glu Val His His Gln LysLeu Val Phe Phe 660 665 670 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala IleIle Gly Leu Met Val 675 680 685 Gly Gly Val Val Ile Ala Thr Val Ile ValIle Thr Leu Val Met Leu 690 695 700 Lys Lys Lys Gln Tyr Thr Ser Ile HisHis Gly Val Val Glu Val Asp 705 710 715 720 Ala Ala Val Thr Pro Glu GluArg His Leu Ser Lys Met Gln Gln Asn 725 730 735 Gly Tyr Glu Asn Pro ThrTyr Lys Phe Phe Glu Gln Met Gln Asn Lys 740 745 750 Lys 62 8 PRTArtificial sequence Synthetic peptide 62 Leu Glu Val Leu Phe Gln Gly Pro1 5 63 10 PRT Artificial sequence Synthetic peptide 63 Ser Glu Val AsnLeu Asp Ala Glu Phe Arg 1 5 10 64 10 PRT Artificial sequence Syntheticpeptide 64 Ser Glu Val Lys Met Asp Ala Glu Phe Arg 1 5 10 65 15 PRTArtificial sequence Synthetic peptide 65 Arg Arg Gly Gly Val Val Ile AlaThr Val Ile Val Gly Glu Arg 1 5 10 15 66 4 PRT Artificial sequenceSynthetic peptide 66 Asn Leu Asp Ala 1 67 8 PRT Artificial sequencePeptide 67 Glu Val Lys Met Asp Ala Glu Phe 1 5 68 5 PRT Artificialsequence peptide 68 Gly Arg Arg Gly Ser 1 5 69 6 PRT Artificial sequencepeptide 69 Thr Gln His Gly Ile Arg 1 5 70 6 PRT Artificial sequencepeptide 70 Glu Thr Asp Glu Glu Pro 1 5 71 15 PRT Artificial sequencepeptide 71 Met Cys Ala Glu Val Lys Met Asp Ala Glu Phe Lys Asp Asn Pro 15 10 15 72 4 PRT Artificial sequence Peptide of Human APP 72 Xaa Xaa XaaXaa 1 73 476 PRT Mus musculus 73 Met Ala Pro Ala Leu His Trp Leu Leu LeuTrp Val Gly Ser Gly Met 1 5 10 15 Leu Pro Ala Gln Gly Thr His Leu GlyIle Arg Leu Pro Leu Arg Ser 20 25 30 Gly Leu Ala Gly Pro Pro Leu Gly LeuArg Leu Pro Arg Glu Thr Asp 35 40 45 Glu Glu Ser Glu Glu Pro Gly Arg ArgGly Ser Phe Val Glu Met Val 50 55 60 Asp Asn Leu Arg Gly Lys Ser Gly GlnGly Tyr Tyr Val Glu Met Thr 65 70 75 80 Val Gly Ser Pro Pro Gln Thr LeuAsn Ile Leu Val Asp Thr Gly Ser 85 90 95 Ser Asn Phe Ala Val Gly Ala AlaPro His Pro Phe Leu His Arg Tyr 100 105 110 Tyr Gln Arg Gln Leu Ser SerThr Tyr Arg Asp Leu Arg Lys Gly Val 115 120 125 Tyr Val Pro Tyr Thr GlnGly Lys Trp Glu Gly Glu Leu Gly Thr Asp 130 135 140 Leu Val Ser Ile ProHis Gly Pro Asn Val Thr Val Arg Ala Asn Ile 145 150 155 160 Ala Ala IleThr Glu Ser Asp Lys Phe Phe Ile Asn Gly Ser Asn Trp 165 170 175 Glu GlyIle Leu Gly Leu Ala Tyr Ala Glu Ile Ala Arg Leu Cys Gly 180 185 190 AlaGly Phe Pro Leu Asn Gln Thr Glu Ala Leu Ala Ser Val Gly Gly 195 200 205Ser Met Ile Ile Gly Gly Ile Asp His Ser Leu Tyr Thr Gly Ser Leu 210 215220 Trp Tyr Thr Pro Ile Arg Arg Glu Trp Tyr Tyr Glu Val Ile Ile Val 225230 235 240 Arg Val Glu Ile Asn Gly Gln Asp Leu Lys Met Asp Cys Lys GluTyr 245 250 255 Asn Tyr Asp Lys Ser Ile Val Asp Ser Gly Thr Thr Asn LeuArg Leu 260 265 270 Pro Lys Lys Val Phe Glu Ala Ala Val Lys Ser Ile LysAla Ala Ser 275 280 285 Ser Thr Glu Lys Phe Pro Asp Gly Phe Trp Leu GlyGlu Gln Leu Val 290 295 300 Cys Trp Gln Ala Gly Thr Thr Pro Trp Asn IlePhe Pro Val Ile Ser 305 310 315 320 Leu Tyr Leu Met Gly Glu Val Thr AsnGln Ser Phe Arg Ile Thr Ile 325 330 335 Leu Pro Gln Gln Tyr Leu Arg ProVal Glu Asp Val Ala Thr Ser Gln 340 345 350 Asp Asp Cys Tyr Lys Phe AlaVal Ser Gln Ser Ser Thr Gly Thr Val 355 360 365 Met Gly Ala Val Ile MetGlu Gly Phe Tyr Val Val Phe Asp Arg Ala 370 375 380 Arg Lys Arg Ile GlyPhe Ala Val Ser Ala Cys His Val His Asp Glu 385 390 395 400 Phe Arg ThrAla Ala Val Glu Gly Pro Phe Val Thr Ala Asp Met Glu 405 410 415 Asp CysGly Tyr Asn Ile Pro Gln Thr Asp Glu Ser Thr Leu Met Thr 420 425 430 IleAla Tyr Val Met Ala Ala Ile Cys Ala Leu Phe Met Leu Pro Leu 435 440 445Cys Leu Met Val Cys Gln Trp Arg Cys Leu Arg Cys Leu Arg His Gln 450 455460 His Asp Asp Phe Ala Asp Asp Ile Ser Leu Leu Lys 465 470 475

What is claimed is:
 1. An isolated polynucleotide comprising thenucleotide sequence set forth in SEQ ID NO:
 7. 2. A vector apolynucleotide according to claim
 1. 3. A vector according to claim 2wherein said polynucleotide is operably linked to a promoter to promoteexpression of the polypeptide encoded by the polynucleotide in a hostcell.
 4. A host cell transformed or transfected with a vector accordingto claim
 3. 5. A host cell according to claim 4 that is a mammaliancell.
 6. A host cell according to claim 4 that expresses the polypeptideon its surface.
 7. A host cell according to claim 4, wherein the hostcell is transfected with a nucleic acid comprising a nucleotide sequencethat encodes an amyloid precursor protein (APP) that includes twocarboxy-terminal lysine residues.
 8. A host cell according to claim 7that expresses the polypeptide and the APP on its surface.
 9. A methodof making a murine Asp2 polypeptide comprising steps of culturing a hostcell of claim 4 in a culture medium under conditions in which the cellproduces the polypeptide that is encoded by the polynucleotide.
 10. Amethod according to claim 9, further comprising a step of purifying thepolypeptide from the cell or the culture medium.
 11. A host celltransformed or transfected with a polynucleotide according to claim 1.12. A host cell according to claim 11 that is a mammalian cell.
 13. Ahost cell according to claim 11 that expresses the polypeptide on itssurface.
 14. A host cell according to claim 11, wherein the host cell istransfected with a nucleic acid comprising a nucleotide sequence thatencodes an amyloid precursor protein (APP) or fragment thereofcontaining a β-secretase cleavage site.
 15. A host cell according toclaim 14 wherein the APP includes two carboxy-terminal lysine residues.16. A host cell according to claim 14 wherein the APP comprises theSwedish mutation (K→N, M→L) adjacent to the β-secretase cleavage site.17. A host cell according to claim 14 that expresses the polypeptide andthe APP on its surface.
 18. A method of making a murine Asp2 polypeptidecomprising steps of culturing a host cell of claim 11 in a culturemedium under conditions in which the cell produces the polypeptide thatis encoded by the polynucleotide.
 19. A method according to claim 18,further comprising a step of purifying the polypeptide from the cell orthe culture medium.